Liquid crystal based analyte detection

ABSTRACT

The present invention relates to the field of detection of viruses, and in particular to detection of viruses using a liquid crystal assay format. In the present invention, virus binding in a detection region is identified by changes in liquid crystal orientation caused by virus binding independent orientation caused by any topography associated with the detection region.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/897,626, filed Jul. 23, 2004, which claims the benefit of U.S.Provisional Application 60/490,122, filed Jul. 25, 2003; U.S.Provisional Application 60/518,706, filed Nov. 10, 2003; and U.S.Provisional Application 60/541,516, filed Feb. 3, 2004, all of which areherein incorporated by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under SBIR Grant No.5R43AI4960602 awarded by the National Institutes of Health/NIAID. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of detection of analytes, andin particular to detection of viruses, cells, bacteria, lipid-membranecontaining organisms, proteins, nucleic acids, carbohydrates and otherbiomolecules, organic molecules and inorganic molecules using a liquidcrystal assay format.

BACKGROUND OF THE INVENTION

The detection of pathogen, protein, and nucleic acid targets inbiological samples forms the basis of the multi-billion dollar in vitrodiagnostic industry. Detection of protein and nucleic acid targets canbe divided into diagnostic and research based markets. The diagnosticmarket includes the detection and identification of pathogens such asviruses and bacteria, the identification of various genetic markers, andthe identification of markers associated with the presence of tumors.The research market includes the genomics and proteomics industries,which require analytical, drug discovery, and high-throughput screeningtechnologies.

Initial viral diagnostics consisted of the crude, albeit sensitive andnon-specific techniques of direct inoculation of sample material intosuckling mice, embryonated eggs, or living cells. Diagnostic methodshave since evolved to the sensitive, specific, but time consumingserological techniques of neutralization, ELISA and fluorescent antibodyassays and subsequently to the current highly sensitive,instrumentation-dependent techniques of nucleic acid amplification andluminescent bead-based assays. This evolution in approach to virusdetection and identification has been driven by advances in biology(cell culture, immunology), followed by advances in biochemistry(immunochemistry, molecular biology, dye chemistry). More recentprogress comes from advances in instrumentation sciences (optics,electronics, robotics, miniaturization, microfluidics, etc.) and by thesubsequent interfacing of microelectronics with biology to develop thefirst generation of biosensors.

There are many ways to detect the presence of a virus in a sample.Methods with the highest sensitivity (real-time PCR, tissue culture,electron microcopy) also involve the highest complexity and/or cost,require sophisticated equipment and facilities and require highlytrained personnel. Methods with less sensitivity (IFA, ELISA, dipstickmethods), in practice, suffer from cross-reactivity problems, involvemore hands-on time and/or are less adaptable to rapidly screening largenumbers of samples. There is a great need for multiplexing in situationssuch as arbovirus surveillance, bio-threat monitoring, and for rapidagent identification during a disease outbreak of unknown origin. Inpractice, nucleic acid techniques and bead-based techniques currentlycan multiplex approximately 6-20 different targets.

Though there are many techniques available to detect and identifyviruses, there is need for improvement. Among the desired attributesare: lower cost, less reliance on biological systems, less reliance onuse of labile, expensive reagents, less complexity in execution,decreased hands-on time required for processing the sample and executionof the assay, minimal technical proficiency for running assays andinterpreting results, miniaturization and portability of equipment,automation, and an increase in multiplexing capability.

SUMMARY OF THE INVENTION

The present invention relates to the field of detection of analytes, andin particular to detection of viruses, cells, bacteria, lipid-membranecontaining organisms, proteins, nucleic acids, carbohydrates and otherbiomolecules, organic molecules and inorganic molecules using a liquidcrystal assay format. Accordingly, the present invention providesmethods for detecting viruses comprising: a) providing: i) a samplesuspected of containing of a virus; ii) a detection device comprising asubstrate comprising at least one detection region having a first virusrecognition moiety immobilized thereon; and iii) mesogens; b) contactingsaid detection region with said sample; and c) contacting said substratewith said mesogens, wherein the presence of said virus is indicated by achange in said mesogens over said detection regions and wherein saidchange is independent of the presence of an additional homeotropicdirector on said detection region. The present invention is not limitedto the detection of any particular change in the mesogens forming theliquid crystal. Indeed, a variety of changes may be detected, including,but not limited to a change in color, a change in texture, a change intilt, and homeotropic orientation.

The present invention is not limited to the detection of any particulartype of virus. Indeed, the detection of a variety of viruses iscontemplated, including, but not limited to viruses in the followingfamilies: Adenoviridae, Arenaviridae, Astroviridae, Birnaviridae,Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Filoviridae,Flaviviridae, Hepadnaviridae, Herpesviridae, Iridoviridae, Filoviridae,Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae,Picornaviridae, Poxyiridae, Reoviridae, Retroviridae, Rhabdoviridae,Togaviridae, Badnavirus, Bromoviridae, Comoviridae, Geminiviridae,Partitiviridae, Potyviridae, Sequiviridae, and Tombusviridae. In someembodiments, the virus is a Japanese Encephalitis Virus group virus. Inother embodiments, the Japanese Encephalitis Virus group virus isselected from the group consisting of West Nile Virus and St. LouisEncephalitis Virus. In still further embodiments, the virus is anenveloped virus.

The present invention is not limited to the use of any particularsubstrate. Indeed, the use of a variety of substrates is contemplated,including, but not limited to metal films, glass, silicon, diamond andpolymeric materials. The present invention is not limited to the use ofany particular polymeric materials. Indeed, the use of a variety ofpolymeric materials is contemplated, including, but not limited to thoseselected from the group consisting of polyurethane, PDMS, polyimide,polystyrene, polycarbonate and polyisocyanoacrylate. The presentinvention is not limited to the use of any particular mesogen. Indeed,the use of a variety of mesogens is contemplated, including, but notlimited to those selected from the group consisting of4-cyano-4′-pentylbiphenyl, N-(4-methoxybenzylidene)-4-butlyaniline andcombinations thereof. The present invention is not limited to the use ofany particular virus recognition moiety. Indeed, the use of a variety ofvirus recognition moieties is contemplated, including, but not limitedto antigen binding proteins and nucleic acids. In some embodiments, theantigen binding protein is an immunoglobulin.

In some embodiments, the substrate comprises a plurality of detectionregions. In some embodiments, the plurality of detection regions havethe same virus recognition moiety bound thereto. In other embodiments,the plurality of detection regions have different virus recognitionmoieties bound thereto. In some embodiments, the detection devicefurther comprises a second substrate arranged opposite said firstsubstrate to form a cell.

In still further embodiments, the change in the mesogens is detected byviewing said detection device between cross polar lenses. In someembodiments, the detection region does not homeotropically orientmesogens in the absence of virus. In some embodiments, homeotropicordering is observed within 48 hours of the application of said sampleto said detection region.

The present invention is not limited to the analysis of any particulartype of sample. Indeed, the analysis of a variety of samples iscontemplated, including, but not limited to biological fluids, tissuehomogenates, feces, vesicular fluids, swabs of orifices or tissues, andmedia in which virus has been cultured or prepared. The presentinvention is not limited to the analysis of any particular type ofbiological fluid. Indeed, the present invention contemplates theanalysis of a variety of biological fluids, including, but not limitedto cerebral-spinal fluid, urine, serum, plasma, nasal secretions,sputum, semen and saliva.

In some embodiments, the present invention provides devices for thedetection of a virus comprising a first substrate comprising at leastone detection region having a first virus recognition moiety specificfor said virus immobilized thereon, wherein said detection region doesnot homeotropically orient an added mesogen in the absence of saidvirus. In some embodiments, the first substrate comprises a plurality ofdetection regions. The present invention is not limited to the use ofany particular substrate. Indeed, the use of a variety of substrates iscontemplated, including, but not limited to metal films, glass, silicon,diamond and polymeric materials. The present invention is not limited tothe use of any particular polymeric materials. Indeed, the use of avariety of polymeric materials is contemplated, including, but notlimited to those selected from the group consisting of polyurethane,PDMS, polyimide, polystyrene, polycarbonate and polyisocyanoacrylate.

The present invention is not limited to the use of any particular virusrecognition moiety. Indeed, the use of a variety of virus recognitionmoieties is contemplated, including, but not limited to antigen bindingproteins and nucleic acids. In some embodiments, the antigen bindingprotein is an immunoglobulin. In some embodiments, the substratecomprises a plurality of detection regions. In some embodiments, theplurality of detection regions have the same virus recognition moietybound thereto. In other embodiments, the plurality of detection regionshave different virus recognition moieties bound thereto. In someembodiments, the detection device further comprises a second substratearranged opposite said first substrate to form a cell. In still furtherembodiments, the plurality of detection regions are arranged in anarray. In some embodiments, the substrates further comprise at least onecontrol region comprising immobilized virus. In other embodiments, thedevice comprises a second substrate oriented opposite said firstsubstrate to form a cell for containing mesogens. In still otherembodiments, the devices comprise cross polar lenses oriented on eitherside of said first substrate and said substrate. In some embodiments,the substrate comprises microchannels in said first substrate, whereinsaid microchannels deliver sample to said detection region.

In still further embodiments, the present invention provides a kitcomprising: a) a device for the detection of a virus comprising a firstsubstrate comprising at least one detection region having a first virusrecognition moiety specific for said virus immobilized thereon, whereinsaid detection region does not homeotropically orient an added mesogenin the absence of said virus; and b) instructions for detection of saidvirus. In some embodiments, the kit further comprises a vial containingmesogens. In other embodiments, the kit further comprises a vialcontaining a virus for use as a positive control.

In still further embodiments, the present invention provides methodscomprising: a) providing a functionalized detection substrate treated toalign mesogens, a stamp substrate displaying at least one ligand, abiological test sample suspected of containing a binding partner for theligand, and mesogens; b) contacting the test sample with the stampsubstrate under conditions such that the binding partner can bind theligand; c) contacting the detection substrate with the stamp substrateunder conditions such that the binding partner to the ligand istransferred to the detection substrate; d) detecting the presence of thebinding partner to the ligand on the detection substrate by applying themesogens to the substrate. The present invention is not limited to usewith any particular biological sample. Indeed, the use of a variety ofbiological samples is contemplated, including, but not limited to, thoseselected from the group consisting of whole blood, serum, cerebralspinal fluid, nasopharyngeal aspirate, and nasal secretions. In someembodiments, the alignment of the mesogens by the detection substrate isdisrupted by the presence of the binding partner to the ligand. In somepreferred embodiments, the alignment is homeotropic. In furtherembodiments, the mesogens are not homeotropically aligned over areas ofthe detection substrate wherein the binding partner of the ligand ispresent. In still other embodiments, the detection substrate is used toform an optical cell. In some embodiments, the detecting is performed byanalysis of the detection substrate with cross-polars. In furtherembodiments, areas of the detection substrate with homeotropicallyaligned mesogens appear dark. In other embodiments, areas of thedetection substrate with substantially non-homeotropically alignedmesogens appear bright. The present invention is not limited to the useof any particular type of ligand. Indeed, the use of a variety ofligands is contemplated. In some embodiments, the ligand is an antigenicsubstance from a pathogenic organism. In some embodiments, the antigenicsubstance is a protein. In further embodiments, the protein is anenvelope protein of a virus. In some preferred embodiments, the envelopeprotein is protein E from West Nile Virus. The present invention is notlimited to the detection of any particular type of binding partner.Indeed, the detection of a variety of binding partners is contemplated.In some embodiments, the binding partner is an antibody. The presentinvention is not limited to the use of any particular number of ligandson a particular substrate. In some embodiments, the stamp substratecomprises two or more ligands in an array. In some preferredembodiments, the ligand is bound by binding partners from a plurality ofspecies or genera. The present invention is not limited to the use ofany particular mesogen. Indeed, the use of a variety of mesogens iscontemplated. In some preferred embodiments, the mesogen is 5CB. Thepresent invention is not limited to the use of any particular type ofmaterial to make the stamp substrate. Indeed, the use of a variety ofmaterials is contemplated. In some preferred embodiments, the stampsubstrate comprises PDMS. The present invention is not limited to theuse of any particular type of detection substrate. Indeed, the use of avariety of test substrates is contemplated. In some preferredembodiments, the detection substrate comprises obliquely deposited gold.

In still further embodiments, the present invention provides kitscomprising: a) a stamp substrate displaying at least one ligand; b) afunctionalized detection substrate that orients mesogens; and c)instructions for using the substrates for detecting a binding partner ofthe ligand. In some embodiments, the kits further comprise a containerof mesogens. The present invention is not limited to the use of anyparticular mesogen. Indeed, the use of a variety of mesogens iscontemplated. In some preferred embodiments, the mesogen is 5CB. Thepresent invention is not limited to the use of any particular type ofmaterial to make the stamp substrate. Indeed, the use of a variety ofmaterials is contemplated. In some preferred embodiments, the stampsubstrate comprises PDMS. The present invention is not limited to theuse of any particular type of detection substrate. Indeed, the use of avariety of test substrates is contemplated. In some preferredembodiments, the detection substrate comprises obliquely deposited gold.The present invention is not limited to the use of any particular typeof ligand. Indeed, the use of a variety of ligands is contemplated. Insome embodiments, the ligand is an antigenic substance from a pathogenicorganism. In some embodiments, the antigenic substance is a protein. Infurther embodiments, the protein is an envelope protein of a virus. Insome preferred embodiments, the envelope protein is protein E from WestNile Virus. In some embodiments, the kits further comprise a containercontaining a control binding partner. The present invention is notlimited to any particular binding partner. Indeed, a variety of bindingpartners are contemplated. In some embodiments, the binding partner is aligand. In some embodiments, the kits further comprise an additionalsubstrate for forming an optical cell. In still other embodiments, thekits comprise polarized lenses.

In still further embodiments, the present invention provides systems fordetecting an analyte comprising: a) a first substrate displaying arecognition moiety, wherein the recognition moiety interacts with theanalyte; b) a second substrate comprising a surface configured toreceive the analyte interacting with the recognition moiety; and c) aliquid crystal overlaying the second substrate. In some embodiments, thefirst substrate is selected from the group consisting of a stamp, abead, and column media. In some embodiments, the stamp comprises PDMS.In some embodiments, the bead is a magnetic bead. In some embodiments,the column is immunoaffinity column media. In some embodiments, therecognition moiety is selected from the group consisting of a protein,polypeptide, peptide, nucleic acid, carbohydrate, lipid, organicmolecule and inorganic molecule. In some embodiments, the liquid crystalcomprises mesogens selected from the group consisting of E7, MLC, 5CB(4-n-pentyl-4′-cyanobiphenyl), 8CB (4-cyano-4′octylbiphenyl), BL093, TL216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline). In some embodiments, the secondsubstrate comprises a functionalized surface. In some embodiments, thefunctionalized surface comprises a polyimide. In some embodiments, thepolyimide is rubbed. In some embodiments, the polyimide is selected fromthe group consisting of Nissan 7210, Nissan 3510, Nissan 410, Nissan3140, Nissan 5291, and Japan Synthetic Rubber JALS 146-R19. In someembodiments, the polyimide homeotropically orients the liquid crystal.In some embodiments, the polyimide is selected from the group consistingof Nissan 7511L and SE 1211.

In further embodiments, the present invention provides methods ofdetecting an analyte comprising: a) providing a first substratedisplaying a recognition moiety, a second substrate, mesogens, and asample suspected of containing an analyte; b) contacting the firstsubstrate displaying a recognition moiety with the sample suspected ofcontaining an analyte so that the analyte interacts with the recognitionmoiety; c) transferring the analyte interacting with the recognitionmoiety to the second substrate; and d) contacting the second substratewith the mesogens to detect the presence of the analyte on the secondsubstrate. In some embodiments, the recognition moiety is selected fromthe group consisting of a protein, polypeptide, peptide, nucleic acid,carbohydrate, lipid, organic molecule and inorganic molecule. In someembodiments, the analyte is selected from the group consisting of aprotein, polypeptide, peptide, nucleic acid, organic molecule, inorganicmolecule, virus, liposome, bacteria, fungus, and cell. In someembodiments, the first substrate is selected from the group consistingof a stamp, a bead, and column media. In some embodiments, the secondsubstrate is selected from the group consisting of silicon, glass,polymer, diamond, and metal. In some embodiments, the second substratecomprises a surface functionalized with a polyimide. In someembodiments, the polyimide is rubbed. In some embodiments, the polyimideis selected from the group consisting of Nissan 7210, Nissan 3510,Nissan 410, Nissan 3140, Nissan 5291, and Japan Synthetic Rubber JALS146-R19. In some embodiments, the polyimide homeotropically orients theliquid crystal. In some embodiments, the polyimide is selected from thegroup consisting of Nissan 7511L and SE 1211. In some embodiments, thepresence of analyte is indicated by a non-ordered liquid crystal thatappears white or bright when viewed through cross polar lenses and areaswhere analyte is not bound remain ordered and appear dark when viewedthrough cross polar lenses. In some embodiments, the presence of ananalyte is indicated by a disordered liquid crystal that appears whiteor bright when viewed through cross polar lenses and areas where noanalyte is bound maintain homeotropic orientation and appear dark. Insome embodiments, the mesogens are selected from the group consisting ofE7, MLC, 5CB (4-n-pentyl-4′-cyanobiphenyl), 8CB(4-cyano-4′octylbiphenyl), BL093, TL 216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline). In some embodiments, thepresence of an analyte on the second substrate is indicated by adifference in the orientation of the mesogens. In some embodiments, thedifference in the orientation of the mesogens is detected by a methodselected from the group consisting of visual detection, opticaldetection, spectroscopy, light transmission, and electrical detection.In some embodiments, the transferring step further comprises the step ofeluting the analyte from the first substrate. In some embodiments, themethods further comprise the step of contacting the analyte-recognitionmoiety complex with a secondary binding agent. In some embodiments, thesecondary binding agent is selected from the group consisting of anantigen binding protein, and enzyme, avidin, and biotin. In someembodiments, the presence of the secondary binding agent enhances thedetection of the analyte after transfer to the second substrate. In someembodiments, the secondary binding agent is complexed with a lipid. Insome embodiments, the secondary binding agent is displayed on aliposome.

In still further embodiments, the present invention provides kitscomprising a) a first substrate displaying a recognition moiety, whereinthe recognition moiety interacts with an analyte; b) a second substratecomprising a surface configured to receive the analyte interacting withthe recognition moiety; c) a vial containing mesogens; and d)instructions for detecting the analyte. In some embodiments, the firstsubstrate is selected from the group consisting of a stamp, a bead, andcolumn media. In some embodiments, the stamp comprises PDMS. In someembodiments, the bead is a magnetic bead. In some embodiments, thecolumn is an immunoaffinity column. In some embodiments, the recognitionmoiety is selected from the group consisting of a protein, polypeptide,peptide, nucleic acid, carbohydrate, lipid, organic molecule andinorganic molecule. In some embodiments, the mesogens are selected fromthe group consisting of E7, MLC, 5CB (4-n-pentyl-4′-cyanobiphenyl), 8CB(4-cyano-4′octylbiphenyl), BL093, TL 216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline). In some embodiments, the secondsubstrate comprises a functionalized surface. In some embodiments, thefunctionalized surface comprises a polyimide. In some embodiments, thepolyimide is rubbed. In some embodiments, the polyimide is selected fromthe group consisting of Nissan 7210, Nissan 3510, Nissan 410, Nissan3140, Nissan 5291, and Japan Synthetic Rubber JALS146-R19. In someembodiments, the polyimide homeotropically orients the liquid crystal.In some embodiments, the polyimide is selected from the group consistingof Nissan 7511L and SE 1211. In some embodiments, the kits furthercomprise a second vial comprising a secondary binding agent. In someembodiments, the secondary binding agent is selected from the groupconsisting of an antigen binding protein, an enzyme, avidin and biotin.

In still further embodiments, the present invention provides methods fordetecting analytes comprising: a) providing: i) a sample suspected ofcontaining of an analyte; ii) a detection device comprising a substratecomprising at least one electrode and at least one detection region;iii) mesogens; b) applying an electrical potential to the at least oneelectrode to transport the analyte to the substrate; and, c) contactingthe substrate with the mesogens, wherein the presence of the analyte isindicated by a difference in alignment of the mesogens over the at leastone detection region. In some embodiments, the electrical potential isan alternating current. In some embodiments, the transport occurs viadielectrophoresis. In some embodiments, the difference in the alignmentof the mesogens is selected from the group consisting of a change incolor, a change in texture, a change in tilt, and homeotropicorientation. In some embodiments, the difference in alignment of themesogens is detected by a method selected from the group consisting ofvisual detection, optical detection, spectroscopy, light transmission,and electrical detection. In some embodiments, the analyte is selectedfrom the group consisting of a protein, peptide, polypeptide, nucleicacid, organic molecule, inorganic molecule, virus, bacteria, liposome,cell, and fungus. In some embodiments, the substrate is selected fromthe group consisting of metal films, glass, silicon, diamond andpolymeric materials. In some embodiments, the polymeric materials areselected from the group consisting of polyurethane, PDMS, polyimide,polystyrene, polycarbonate and polyisocyanoacrylate. In someembodiments, the mesogen is selected from the group consisting of E7,MLC, 5CB (4-n-pentyl-4′-cyanobiphenyl), 8CB (4-cyano-4′octylbiphenyl),BL093, TL 216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline) and combinations thereof. Insome embodiments, the detection region comprises a recognition moiety.In some embodiments, the recognition moiety is selected from the groupconsisting of an peptide, polypeptide, protein, nucleic acid,carbohydrate, organic molecule, and inorganic molecule. In someembodiments, the protein is an antigen binding protein. In someembodiments, the substrate comprises a plurality of detection regions.In some embodiments, the plurality of detection regions display the samerecognition moiety. In some embodiments, the plurality of detectionregions display different recognition moieties. In some embodiments, thedetection device further comprises a second substrate arranged oppositethe first substrate to form a cell. In some embodiments, the change inthe mesogens is detected by viewing the detection device between crosspolar lenses. In some embodiments, the sample is selected from the groupconsisting of biological fluids, tissue homogenates, feces, vesicularfluids, swabs of orifices or tissues, and media in which virus has beencultured or prepared. In some embodiments, the biological fluid isselected from the group consisting of cerebral-spinal fluid, urine,serum, plasma, nasal secretions, sputum, semen and saliva. In someembodiments, the methods further comprise the step of detecting analytebinding by measuring the impedance of the detection device, wherein achange in capacitance is indicative of analyte binding. In someembodiments, the impedance is capacitance or resistance. In someembodiments, the measuring is in real-time.

In still further embodiments, the present invention provides devices fordetecting an analyte comprising a first substrate comprising at leastone electrode and at least one detection region, wherein the at leastone electrode is configured to provide an electrical potential toattract an analyte to the substrate and to determine the presence of theanalyte by measuring electrical properties of the device, and a secondsubstrate oriented opposite of the first substrate, wherein the firstsubstrate and the second substrate form a chamber for containing aliquid crystal. In some embodiments, the electrical property isimpedance. In some embodiments, the impedance is capacitance orresistance. In some embodiments, the impedance is capacitance. In someembodiments, the substrate is selected from the group consisting ofmetal films, glass, silicon, diamond and polymeric materials. In someembodiments, the polymeric materials are selected from the groupconsisting of polyurethane, PDMS, polyimide, polystyrene, polycarbonateand polyisocyanoacrylate. In some embodiments, the devices furthercomprise mesogens, wherein the mesogens are selected from the groupconsisting of E7, MLC, 5CB (4-n-pentyl-4′-cyanobiphenyl), 8CB(4-cyano-4′octylbiphenyl), BL093, TL 216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline) and combinations thereof. Insome embodiments, the detection region comprises a recognition moiety.In some embodiments, the recognition moiety is selected from the groupconsisting of a peptide, polypeptide, protein, nucleic acid,carbohydrate, organic molecule, and inorganic molecule. In someembodiments, the protein is an antigen binding protein. In someembodiments, the first substrate comprises a plurality of detectionregions. In some embodiments, the plurality of detection regions displaythe same recognition moiety. In some embodiments, the plurality ofdetection regions display different recognition moieties. In someembodiments, the at least one electrode is selected from the groupconsisting of interdigitated, hyperbolic, triangular and rectangularelectrodes. In some embodiments, the first substrate comprises at leasttwo electrodes.

In still further embodiments, the present invention provides systems fordetection of an analyte comprising the detection device described aboveand a readout device, the readout device comprising an openingconfigured to receive the detection device and an electrical circuitthat contacts the at least one electrode when the detection device is incontact with the readout device. In some embodiments, the readout deviceinterfaces with a computer processor. In some embodiments, the readoutdevice comprises an electronic display. In some embodiments, the readoutdevice comprises an LCD display. In some embodiments, the electriccircuit is an oscillator circuit. In some embodiments, the oscillatorcircuit comprises a microprocessor. In some embodiments, the readoutdevice comprises a microprocessor configured to measure electricalcapacitance. In some embodiments, the readout device comprises a powersource.

In still other embodiments, the present invention provides methods ofdetecting an analyte comprising: a) providing a sample suspected ofcontaining an analyte, a substrate having a surface comprisingpolyimide, and mesogens; b) contacting the surface comprising polyimidewith the sample suspected of containing an analyte; c) contacting thesurface comprising polyimide with the mesogens, wherein the presence ofthe analyte is indicated by difference in the orientation of themesogens. In some embodiments, the analyte non-specifically interactswith the surface comprising polyimide. In some embodiments, the surfacecomprising polyimide displays a recognition moiety. In some embodiments,the recognition moiety is selected from the group consisting of aprotein, polypeptide, peptide, nucleic acid, carbohydrate, lipid,organic molecule and inorganic molecule. In some embodiments, themesogens are selected from the group consisting of E7, MLC, 5CB(4-n-pentyl-4′-cyanobiphenyl), 8CB (4-cyano-4′octylbiphenyl), BL093, TL216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline). In some embodiments, thepolyimide is rubbed. In some embodiments, the polyimide is selected fromthe group consisting of Nissan 7210, Nissan 3510, Nissan 410, Nissan3140, Nissan 5291, and Japan Synthetic Rubber JALS146-R19. In someembodiments, the polyimide homeotropically orients the mesogens. In someembodiments, the polyimide is selected from the group consisting ofNissan 7511L and SE 1211. In some embodiments, the presence of analyteis indicated by a disordered liquid crystal that appears white or brightwhen viewed through cross polar lenses and areas where analyte is notbound remain ordered and appear dark when viewed through cross polarlenses. In some embodiments, the presence of an analyte is indicated bya disordered liquid crystal that appears white or bright when viewedthrough cross polar lenses and areas where no analyte is bound maintainhomeotropic orientation and appear dark. In some embodiments, thehomeotropic orientation of the liquid crystal is detected by a methodselected from the group consisting of visual detection, opticaldetection, spectroscopy, light transmission, and electrical detection.

In some embodiments, the present invention provides methods of detectingbinding interaction between a ligand and its binding partner comprising:a) providing a ligand and a binding partner, wherein at least one of theligand molecule and the binding partner molecule are complexed with alipid, and mesogens; b) contacting the ligand molecule and the bindingpartner molecule under conditions such that the ligand molecule and thebinding partner molecule interact to form a ligand-binding partnercomplex; and c) detecting the ligand-binding partner complex bycontacting the complex with mesogens. In some embodiments, the mesogensare homeotropically oriented by the complex. In some embodiments, thebinding partner is recognition moiety. In some embodiments, the ligandis an analyte a sample. In some embodiments, the detecting step furthercomprises contacting the complex to a substrate prior to contacting withthe mesogens. In some embodiments, the homeotropic alignment of themesogens is detected by a method selected from the group consisting ofvisual detection, optical detection, spectroscopy, light transmission,and electrical detection. In some embodiments, the analyte is selectedfrom the group consisting of a protein, peptide, polypeptide, nucleicacid, organic molecule, inorganic molecule, virus, bacteria, liposome,cell, and fungus. In some embodiments, the substrate is selected fromthe group consisting of metal films, glass, silicon, diamond andpolymeric materials. In some embodiments, the polymeric materials areselected from the group consisting of polyurethane, PDMS, polyimide,polystyrene, polycarbonate and polyisocyanoacrylate. In someembodiments, the mesogen is selected from the group consisting of E7,MLC, 5CB (4-n-pentyl-4′-cyanobiphenyl), 8CB (4-cyano-4′octylbiphenyl),BL093, TL 216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline) and combinations thereof. Insome embodiments, the substrate comprises a detection region comprisinga recognition moiety. In some embodiments, the recognition moiety isselected from the group consisting of a peptide, polypeptide, protein,nucleic acid, carbohydrate, organic molecule, and inorganic molecule. Insome embodiments, the protein is an antigen binding protein. In someembodiments, the substrate comprises a plurality of detection regions.In some embodiments, the plurality of detection regions display the samerecognition moiety. In some embodiments, the plurality of detectionregions display different recognition moieties. In some embodiments, theligand id biotin and the recognition moiety is avidin. In someembodiments, the at least one of the ligand molecule and the bindingpartner molecule complexed with a lipid is a secondary binding agent.

In some embodiments, the present invention provides kits for detectingan analyte comprising: a) a recognition moiety complexed with a lipid;b) a vial containing mesogens; and c) instructions for detecting theanalyte. In some embodiments, the kits further comprise a substrate. Insome embodiments, the recognition moiety is selected from the groupconsisting of a protein, polypeptide, peptide, nucleic acid,carbohydrate, organic molecule and inorganic molecule. In someembodiments, the substrate is selected from the group consisting ofsilicon, glass, polymer, diamond, and metal. In some embodiments, thesubstrate does not orient the liquid crystal. In some embodiments, themesogens are selected from the group consisting of E7, MLC, 5CB(4-n-pentyl-4′-cyanobiphenyl), 8CB (4-cyano-4′octylbiphenyl), BL093, TL216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline). In some embodiments, thepresent invention provides systems for detecting an analyte comprising:a) a recognition moiety complexed with a lipid; and b) a liquid crystal.In some embodiments, the systems further comprise a substrate. In someembodiments, the recognition moiety is selected from the groupconsisting of a protein, polypeptide, peptide, nucleic acid,carbohydrate, organic molecule and inorganic molecule. In someembodiments, the substrate is selected from the group consisting ofsilicon, glass, polymer, diamond, and metal. In some embodiments, thesubstrate does not orient the liquid crystal. In some embodiments, themesogens are selected from the group consisting of E7, MLC, 5CB(4-n-pentyl-4′-cyanobiphenyl), 8CB (4-cyano-4′octylbiphenyl), BL093, TL216, ZLI 5800, MLC 6613, and MBBA((p-methoxybenzylidene)-p-butylaniline).

DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic view of an assay device of the presentinvention demonstrating homeotropic orientation of a liquid crystaldirected by bound virus.

FIG. 2 provides photographs of transfers assays for the presence of WestNile Virus antibodies in positive horse and rabbit serum along withnegative controls.

FIGS. 3 a and 3 b provide a schematic depiction of a device andpreferred electrodes of the present invention.

FIG. 4 is an image of a PDMS stamp of the present invention.

FIG. 5 is an image of an assay conducted with an assay device of thepresent invention.

FIG. 6 shows the results from a simulation of hyperbolic electrodes.

FIG. 7 shows a schematic of an device configured for dielectrophoresis.

FIG. 8 is an image of an assay using a polyimide coated substrate tonon-specifically detect an analyte.

FIG. 9 is an image of an assay using a polyimide coated substrate tonon-specifically detect an analyte.

FIG. 10 is a graphic representation of luminosity index for theexperiment depicted in FIG. 9.

FIG. 11 is an image of an assay using a polyimide coated substrate tonon-specifically detect an analyte.

FIG. 12 is an image of the results of the detection of F1 in chickenserum.

FIG. 13 is an image of the results of the same experiment as FIG. 12taken with a polarized microscope.

FIG. 14 is a schematic depiction of a readout device of the presentinvention.

FIG. 15 presents images of experiments in which labeled liposomes areused to report ligand binding.

DEFINITIONS

As used herein, the term “recognition moiety” refers to a composition ofmatter that interacts with an analyte of interest in either a covalentor noncovalent manner.

As used herein, the term “virus recognition moiety” refers to anycomposition of matter that binds specifically to a virus. Examples of“virus recognition moieties” include, but are not limited to antigenbinding proteins and nucleic acid aptamers.

The term “substrate” refers to a composition that serves as a base foranother composition such as recognition moiety. Examples of substratesinclude, but are not limited to, silicon surfaces, glass surfaces, glassbeads, magnetic beads, agarose beads, etc.

As used herein, the term “ligand” refers to any molecule that binds toor can be bound by another molecule. A ligand is any ion, molecule,molecular group, or other substance that binds to another entity to forma larger complex. Examples of ligands include, but are not limited to,peptides, carbohydrates, nucleic acids, antibodies, or any moleculesthat bind to receptors.

As used herein, the term “homeotropic director” refers to atopographical feature (e.g., a nanostructure or homeotropic orientingpolyimide) of a substrate that homeotropically orients a liquid crystal.

As used herein, the term “pathogen” refers to disease causing organisms,microorganisms, or agents including, but not limited to, viruses,bacteria, parasites (including, but not limited to, organisms within thephyla Protozoa, Platyhelminthes, Aschelminithes, Acanthocephala, andArthropoda), fungi, and prions.

As used herein, the term “bacteria” and “bacterium” refer to allprokaryotic organisms, including those within all of the phyla in theKingdom Procaryotae. It is intended that the term encompass allmicroorganisms considered to be bacteria including Mycoplasma,Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms ofbacteria are included within this definition including cocci, bacilli,spirochetes, spheroplasts, protoplasts, etc. “Gram negative” and “grampositive” refer to staining patterns obtained with the Gram-stainingprocess which is well known in the art (See e.g., Finegold and Martin,Diagnostic Microbiology, 6th Ed. (1982), CV Mosby St. Louis, pp 13-15).

As used herein, the term “lipid membrane” refers to, in its broadestsense, a thin sheet or layer comprising lipid molecules. It is intendedthat the term encompass all “biomembranes” (i.e., any organic membraneincluding, but not limited to, plasma membranes, nuclear membranes,organelle membranes, and synthetic membranes). Typically, membranes arecomposed of lipids, proteins, glycolipids, steroids, sterol and/or othercomponents. As used herein, the term “membrane fragment” refers to anyportion or piece of a membrane.

As used herein, the term “lipid” refers to a variety of compounds thatare characterized by their solubility in organic solvents. Suchcompounds include, but are not limited to, fats, waxes, steroids,sterols, glycolipids, glycosphingolipids (including gangliosides),phospholipids, terpenes, fat-soluble vitamins, prostaglandins,carotenes, and chlorophylls. As used herein, the phrase “lipid-basedmaterials” refers to any material that contains lipids.

As used herein, the term “liposome” refers to artificially producedspherical lipid complexes that can be induced to segregate out ofaqueous media.

As used herein, the term “secondary binding agent” refer to a moleculeor collection of molecules that binds to one of an analyte-recognitionmoiety complex. It is contemplated that secondary binding agents areuseful for amplifying the signal resulting from analyte-recognitionmoiety binding.

As used herein, the term “column media” refers to media used to fill achromatography column, such as cationic exchange media, anionic exchangemedia, and immunoaffinity column media.

As used herein, the term “detection region” refers to a discreet area onsubstrate that is designated for detection of an analyte (e.g., a virusof interest) in a sample.

As used herein, the term “immobilization” refers to the attachment orentrapment, either chemically or otherwise, of a material to anotherentity (e.g., a solid support) in a manner that restricts the movementof the material.

As used herein, the terms “material” and “materials” refer to, in theirbroadest sense, any composition of matter.

As used herein the term “antigen binding protein” refers to aglycoprotein evoked in an animal by an immunogen (antigen) and toproteins derived from such glycoprotein (e.g., single chain antibodiesand F(ab′)2, Fab′ and Fab fragments). An antibody demonstratesspecificity to the immunogen, or, more specifically, to one or moreepitopes contained in the immunogen. Native antibody comprises at leasttwo light polypeptide chains and at least two heavy polypeptide chains.Each of the heavy and light polypeptide chains contains at the aminoterminal portion of the polypeptide chain a variable region (i.e., VHand VL respectively), which contains a binding domain that interactswith antigen. Each of the heavy and light polypeptide chains alsocomprises a constant region of the polypeptide chains (generally thecarboxy terminal portion) which may mediate the binding of theimmunoglobulin to host tissues or factors influencing various cells ofthe immune system, some phagocytic cells and the first component (C1q)of the classical complement system. The constant region of the lightchains is referred to as the “CL region,” and the constant region of theheavy chain is referred to as the “CH region.” The constant region ofthe heavy chain comprises a CH1 region, a CH2 region, and a CH3 region.A portion of the heavy chain between the CH1 and CH2 regions is referredto as the hinge region (i.e., the “H region”). The constant region ofthe heavy chain of the cell surface form of an antibody furthercomprises a spacer-transmembranal region (M1) and a cytoplasmic region(M2) of the membrane carboxy terminus. The secreted form of an antibodygenerally lacks the M1 and M2 regions.

As used herein, the term “selective binding” refers to the binding ofone material to another in a manner dependent upon the presence of aparticular molecular structure (i.e., specific binding). For example, animmunoglobulin will selectively bind an antigen that contains thechemical structures complementary to the ligand binding site(s) of theimmunoglobulin. This is in contrast to “non-selective binding,” wherebyinteractions are arbitrary and not based on structural compatibilitiesof the molecules.

As used herein, the term “polymerization” encompasses any process thatresults in the conversion of small molecular monomers into largermolecules consisting of repeated units. Typically, polymerizationinvolves chemical crosslinking of monomers to one another.

As used herein, the term “antigen” refers to any molecule or moleculargroup that is recognized by at least one antibody. By definition, anantigen must contain at least one epitope (i.e., the specificbiochemical unit capable of being recognized by the antibody). The term“immunogen” refers to any molecule, compound, or aggregate that inducesthe production of antibodies. By definition, an immunogen must containat least one epitope (i.e., the specific biochemical unit capable ofcausing an immune response).

As used herein, the terms “home testing” and “point of care testing”refer to testing that occurs outside of a laboratory environment. Suchtesting can occur indoors or outdoors at, for example, a privateresidence, a place of business, public or private land, in a vehicle, aswell as at the patient's bedside.

As used herein, the term “virus” refers to minute infectious agents,which with certain exceptions, are not observable by light microscopy,lack independent metabolism, and are able to replicate only within aliving host cell. The individual particles (i.e., virions) consist ofnucleic acid and a protein shell or coat; some virions also have a lipidcontaining membrane. The term “virus” encompasses all types of viruses,including animal, plant, phage, and other viruses.

As used herein, term “nanostructures” refers to microscopic structures,typically measured on a nanometer scale. Such structures include variousthree-dimensional assemblies, including, but not limited to, liposomes,films, multilayers, braided, lamellar, helical, tubular, and fiber-likeshapes, and combinations thereof. Such structures can, in someembodiments, exist as solvated polymers in aggregate forms such as rodsand coils. Such structures can also be formed from inorganic materials,such as prepared by the physical deposition of a gold film onto thesurface of a solid, proteins immobilized on surfaces that have beenmechanically rubbed, and polymeric materials that have been molded orimprinted with topography by using a silicon template prepared byelectron beam lithography.

As used herein, the terms “self-assembling monomers” and “lipidmonomers” refer to molecules that spontaneously associate to formmolecular assemblies. In one sense, this can refer to surfactantmolecules that associate to form surfactant molecular assemblies. Theterm “self-assembling monomers” includes single molecules (e.g., asingle lipid molecule) and small molecular assemblies (e.g., polymerizedlipids), whereby the individual small molecular assemblies can befurther aggregated (e.g., assembled and polymerized) into largermolecular assemblies.

As used herein, the term “linker” or “spacer molecule” refers tomaterial that links one entity to another. In one sense, a molecule ormolecular group can be a linker that is covalent attached two or moreother molecules (e.g., linking a ligand to a self-assembling monomer).

As used herein, the term “bond” refers to the linkage between atoms inmolecules and between ions and molecules in crystals. The term “singlebond” refers to a bond with two electrons occupying the bonding orbital.Single bonds between atoms in molecular notations are represented by asingle line drawn between two atoms (e.g., C—C). The term “double bond”refers to a bond that shares two electron pairs. Double bonds arestronger than single bonds and are more reactive. The term “triple bond”refers to the sharing of three electron pairs. As used herein, the term“ene-yne” refers to alternating double and triple bonds. As used hereinthe terms “amine bond,” “thiol bond,” and “aldehyde bond” refer to anybond formed between an amine group (i.e., a chemical group derived fromammonia by replacement of one or more of its hydrogen atoms byhydrocarbon groups), a thiol group (i.e., sulfur analogs of alcohols),and an aldehyde group (i.e., the chemical group —CHO joined directlyonto another carbon atom), respectively, and another atom or molecule.

As used herein, the term “covalent bond” refers to the linkage of twoatoms by the sharing of two electrons, one contributed by each of theatoms.

As used herein, the term “spectrum” refers to the distribution of lightenergies arranged in order of wavelength.

As used the term “visible spectrum” refers to light radiation thatcontains wavelengths from approximately 360 nm to approximately 800 nm.

As used herein, the term “substrate” refers to a solid object or surfaceupon which another material is layered or attached. Solid supportsinclude, but are not limited to, glass, metals, gels, and filter paper,among others.

As used herein, the terms “array” and “patterned array” refer to anarrangement of elements (i.e., entities) into a material or device. Forexample, combining several types of ligand binding molecules (e.g.,antibodies or nucleic acids) into an analyte-detecting device, wouldconstitute an array.

As used herein, the term “in situ” refers to processes, events, objects,or information that are present or take place within the context oftheir natural environment.

As used herein, the term “sample” is used in its broadest sense. In onesense it can refer to a biopolymeric material. In another sense, it ismeant to include a specimen or culture obtained from any source, as wellas biological and environmental samples. Biological samples may beobtained from animals (including humans) and encompass fluids, solids,tissues, and gases. Biological samples include blood products, such asplasma, serum and the like. Environmental samples include environmentalmaterial such as surface matter, soil, water, crystals and industrialsamples. These examples are not to be construed as limiting the sampletypes applicable to the present invention.

As used herein, the term “liquid crystal” refers to a thermodynamicstable phase characterized by anisotropy of properties without theexistence of a three-dimensional crystal lattice, generally lying in thetemperature range between the solid and isotropic liquid phase.

As used herein, the term “mesogen” refers compounds that form liquidcrystals, and in particular rigid rodlike or dislike molecules that arecomponents of liquid crystalline materials.

As used herein, “thermotropic liquid crystal” refers to liquid crystalsthat result from the melting of mesogenic solids due to an increase intemperature. Both pure substances and mixtures form thermotropic liquidcrystals.

“Lyotropic,” as used herein, refers to molecules that form phases withorientational and/or positional order in a solvent. Lyotropic liquidcrystals can be formed using amphiphilic molecules (e.g., sodiumlaurate, phosphatidylethanolamine, lecithin). The solvent can be water.

As used herein, the term “heterogenous surface” refers to a surface thatorients liquid crystals in at least two separate planes or directions,such as across a gradient.

As used herein, “nematic” refers to liquid crystals in which the longaxes of the molecules remain substantially parallel, but the positionsof the centers of mass are randomly distributed. Nematic liquid crystalscan be substantially oriented by a nearby surface.

“Chiral nematic,” as used herein refers to liquid crystals in which themesogens are optically active. Instead of the director being heldlocally constant as is the case for nematics, the director rotates in ahelical fashion throughout the sample. Chiral nematic crystals show astrong optical activity that is much higher than can be explained on thebases of the rotatory power of the individual mesogens. When light equalin wavelength to the pitch of the director impinges on the liquidcrystal, the director acts like a diffraction grating, reflecting mostand sometimes all of the light incident on it. If white light isincident on such a material, only one color of light is reflected and itis circularly polarized. This phenomenon is known as selectivereflection and is responsible for the iridescent colors produced bychiral nematic crystals.

“Smectic,” as used herein refers to liquid crystals which aredistinguished from “nematics” by the presence of a greater degree ofpositional order in addition to orientational order; the molecules spendmore time in planes and layers than they do between these planes andlayers. “Polar smectic” layers occur when the mesogens have permanentdipole moments. In the smectic A2 phase, for example, successive layersshow anti ferroelectric order, with the direction of the permanentdipole alternating from layer to layer. If the molecule contains apermanent dipole moment transverse to the long molecular axis, then thechiral smectic phase is ferroelectric. A device utilizing this phase canbe intrinsically bistable.

“Frustrated phases,” as used herein, refers to another class of phasesformed by chiral molecules. These phases are not chiral, however, twistis introduced into the phase by an array of grain boundaries. A cubiclattice of defects (where the director is not defined) exist in acomplicated, orientationally ordered twisted structure. The distancebetween these defects is hundreds of nanometers, so these phases reflectlight just as crystals reflect x-rays.

“Discotic phases” are formed from molecules that are disc shaped ratherthan elongated. Usually these molecules have aromatic cores and sixlateral substituents. If the molecules are chiral or a chiral dopant isadded to a discotic liquid crystal, a chiral nematic discotic phase canform.

DESCRIPTION OF THE INVENTION

The present invention relates to the field of detection of analytes, andin particular to detection of viruses, cells, bacteria, lipid-membranecontaining organisms, proteins, nucleic acids, carbohydrates and otherbiomolecules, organic molecules and inorganic molecules using a liquidcrystal assay format. Liquid crystal-based assay systems (LC assays) aredescribed in U.S. Pat. No. 6,284,197; WO 01/61357; WO 01/61325; WO99/63329; Gupta et al., Science 279:2077-2080 (1998); Seung-Ryeol Kim,Rahul R. Shah, and Nicholas L. Abbott; Orientations of Liquid Crystalson Mechanically Rubbed Films of Bovine Serum Albumin: A PossibleSubstrate for Biomolecular Assays Based on Liquid Crystals, AnalyticalChemistry; 2000; 72(19); 4646-4653; Justin J. Skaife and Nicholas L.Abbott; Quantitative Interpretation of the Optical Textures of LiquidCrystals Caused by Specific Binding of Immunoglobulins to Surface-BoundAntigens, Langmuir; 2000; 16(7); 3529-3536; Vinay K. Gupta and NicholasL. Abbott; Using proplets of Nematic Liquid Crystal To Probe theMicroscopic and Mesoscopic Structure of Organic Surfaces, Langmuir;1999; 15(21); 7213-7223; all of which are incorporated herein byreference.

The present invention provides systems, devices, and methods for bothdirect and indirect detection of analytes. The indirect detectionsystems utilize a first substrate comprising a recognition moiety thatinteracts with an analyte of interest, preferably specifically. Afterthe first substrate is exposed to a sample suspected of containing ananalyte, analyte interacting with the recognition moieties displayed onthe first substrate are transferred to the second substrate. Inpreferred embodiments, the analyte interacts with the second substratein a non-specific manner. In further preferred embodiments, the secondsubstrate comprises a detection region that orients mesogens in liquidcrystal. The second substrate is then contacted with a liquid crystal. Adisordered liquid crystal is indicative of the presence of an analyte inthe detection region.

WO 01/61357 describes the detection of viruses using liquid crystalbased assays. These assays utilize a patterned detection region on asubstrate that organizes mesogens in a homeotropic orientation. Theassays are designed so that binding of a virus to the detection regionsdisrupts the homeotropic orientation.

Surprisingly, it has now been discovered that viral particles and otherparticles or organisms and cells having lipid membranes bound to asurface can homeotropically orient mesogens independent of anyunderlying topography pattern on the substrate. Thus, assay devices canbe developed and manufactured without the time consuming and expensivestop of optimizing and fabricating nanostructured surfaces. Homeotropicalignment is observed in the present assays if the lipid membranecontaining entity is either specifically or non-specifically bound to asubstrate surface. The assays of the present invention can utilize avariety of recognition moieties to detect a wide variety of entitieswith lipid membranes in a wide variety of samples. Furthermore, theassays operate independent of temperature constraints. Thus, the assaysof the present invention can be used to detect virtually any entity thatcomprises a lipid membrane that is accessible to mesogens in a liquidcrystal.

In addition to entities with lipids membranes, which are listed below,the devices, systems and methods of the present invention are useful fordetecting a variety of analytes, including, but not limited to, thefollowing analytes: biomolecules including polypeptides (e.g.,proteins), toxins, polynucleotides (e.g., RNA and DNA), carbohydrates,viruses, mycoplasmas, fungi, bacteria, and protozoa, especially Class Aagents such as Variola major (smallpox), Bacillus anthracis (anthrax),Yersinia pestis (plague), Clostridium botulinum (botulism), Francisellatularensis (tularemia), Arenaviruses (Arenaviridae), Ebola hemorrhagicfever virus, Marburg hemorrhagic fever, Lassa fever virus, Junin andrelated viruses (Argentinian hemorrhagic fever virus, Bolivianhemorrhagic fever virus, Brazilian hemorrhagic fever virus, Venezuelanhemorrhagic fever virus), Dengue hemorrhagic fever virus, and toxinssuch as botulinum and Trichothecene (T2) mycotoxins; Class B agents suchas Coxiella burnetti (Q fever), Brucella sp. (brucellosis), Burkholderiamallei (glanders), Salmonella sp., Shigella dysenteria, Escherichia colistrain O 157:H7, Cryptosporidium parvum, Alphaviruses (Togaviridaefamily) such as Venezuelan equine encephalitis virus, Eastern equineencephalitis virus, Western equine encephalitis virus, and toxins suchas ricin toxin, epsilin toxin from Clostridium perfigens, andStaphylococcus enterotoxin B; and Class C agents such as mutlidrugresistant tuberculosis, Nipah virus, Hantaviruses, Tick-bornehemorrhagic fever viruses, Tick-borne encephalitis viruses, and Yellowfever virus. Other analytes include, but are not limited to, acids,bases, organic ions, inorganic ions, pharmaceuticals, herbicides,pesticides, chemical warfare agents, and noxious gases. These agents canbe present as components in mixtures of structurally unrelatedcompounds, racemic mixtures of stereoisomers, non-racemic mixtures ofstereoisomers, mixtures of diastereomers, mixtures of positional isomersor as pure compounds. The detection of these analytes, and specificsubstrates and recognition moieties for such detection, is described inmore detail in co-pending application Ser. Nos. 10/227,974, 10/443,419,and 60/585,275; all of which are incorporated herein by reference intheir entirety.

Accordingly, the present invention provides improved substrates anddevices for the detection of analytes. For convenience, the descriptionof the present invention is divided into the following sections: I.Recognition Moieties; II. Substrates; III. Functionalization ofSubstrates; IV. Mesogens; V. Direct Detection of Entities with LipidMembranes; VI. Non-specific Detection Following Specific Capture; VII.Detection with Lipid Tags VIII. Kits.

I. Recognition Moieties

A variety of recognition moieties find use in the present invention. Inpreferred embodiments, the recognition moieties are immobilized ondetection regions of the substrate (described in more detail below). Insome embodiments of the present invention, a “recognition moiety”attached to or associated with the substrate is utilized to bind to orotherwise interact with another molecule or molecules (e.g., analytes).For example, in some embodiments, recognition moieties are attached toeither co-functionalized spacer arms or co-functionalized SAM componentswhich are in turn attached to or associated with the substrate.Furthermore, a recognition moiety can be presented by a polymer surface(e.g., a rubbed polymer surface).

In some preferred embodiments, the recognition moiety comprises anorganic functional group. In presently preferred embodiments, theorganic functional group is a member selected from the group consistingof amines, carboxylic acids, drugs, chelating agents, crown ethers,cyclodextrins or a combination thereof. In another preferred embodiment,the recognition moiety is a biomolecule. In still further preferredembodiments, the biomolecule is a protein, antigen binding protein,peptide, nucleic acid (e.g., single nucleotides or nucleosides,oligonucleotides, polynucleotides and single- and higher-strandednucleic acids) or a combination thereof. In a presently preferredembodiment, the recognition moiety is biotin. In some embodiments, therecognition moieties are antigen binding proteins. Examples of antigenbinding proteins finding use in the present invention include, but arenot limited to, immunoglobulins, single chain antibodies, chimericantibodies, polyclonal antibodies, monoclonal antibodies, and F(ab′)2,Fab′ and Fab fragments.

Various procedures known in the art may be used for the production ofpolyclonal antibodies. For the production of antibody, various hostanimals, including but not limited to rabbits, mice, rats, sheep, goats,etc., can be immunized by injection with the peptide corresponding to anepitope. In a preferred embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels (e.g., aluminum hydroxide), surface activesubstances (e.g., lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Corynebacterium parvum).

For preparation of monoclonal antibodies, it is contemplated that anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture will find use with the presentinvention (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Theseinclude but are not limited to the hybridoma technique originallydeveloped by Köhler and Milstein (Köhler and Milstein, Nature256:495-497 [1975]), as well as the trioma technique, the human B-cellhybridoma technique (See e.g., Kozbor et al., Immunol. Tod., 4:72[1983]), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 [1985]).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing specific singlechain antibodies that serve as recognition moieties. Furthermore, it iscontemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that are usefulrecognition moieties. For example, such fragments include but are notlimited to: F(ab′)2 fragment that can be produced by pepsin digestion ofthe antibody molecule; Fab′ fragments that can be generated by reducingthe disulfide bridges of the F(ab′)2 fragment, and Fab fragments thatcan be generated by treating the antibody molecule with papain and areducing agent. In still further embodiments, the recognition moietycomprises a phage displaying an antigen binding protein.

In some embodiments where the recognition moiety is a polynucleotide orpolypeptide, a plurality of recognition moieties are arrayed on thesubstrates using photo activated chemistry, microcontact printing, andink-jet printing. In particularly preferred embodiments,photolithography is utilized (See e.g., U.S. Pat. Nos. 6,045,996;5,925,525; and 5,858,659; each of which is herein incorporated byreference). Using a series of photolithographic masks to definesubstrate exposure sites, followed by specific chemical synthesis steps,the process constructs high-density arrays of oligonucleotides, witheach probe in a predefined position in the array. Multiple probe arraysare synthesized simultaneously on, for example, a large glass wafer. Thewafers are then diced, and individual probe arrays are packaged ininjection-molded plastic cartridges, which protect them from theenvironment and serve as chambers for hybridization.

In other embodiments, nucleic acid recognition moieties areelectronically captured on a suitable substrate (See e.g., U.S. Pat.Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics, thistechnology enables the active movement and concentration of chargedmolecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given target areelectronically placed at, or “addressed” to, specific sites on themicrochip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

In still further embodiments, recognition moieties are arrayed on asuitable substrate by utilizing differences in surface tension (Seee.g., U.S. Pat. Nos. 6,001,311; 5,985,551; and 5,474,796; each of whichis herein incorporated by reference). This technology is based on thefact that fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on a X/Y translation stage under aset of four piezoelectric nozzles, one for each of the four standard DNAbases. The translation stage moves along each of the rows of the arrayand the appropriate reagent is delivered to each of the reaction site.For example, the A amidite is delivered only to the sites where amiditeA is to be coupled during that synthesis step and so on. Common reagentsand washes are delivered by flooding the entire surface and thenremoving them by spinning.

In still further embodiments, recognition moieties are spotted onto asuitable substrate. Such spotting can be done by hand with a capillarytube or micropipette, or by an automated spotting apparatus such asthose available from Affymetrix and Gilson (See e.g., U.S. Pat. Nos.5,601,980; 6,242,266; 6,040,193; and 5,700,637; each of which isincorporated herein by reference).

When the recognition moiety is an amine, in preferred embodiments, therecognition moiety will interact with a structure on the analyte whichreacts by binding to the amine (e.g., carbonyl groups, alkylhalogroups). In another preferred embodiment, the amine is protonated by anacidic moiety on the analyte of interest (e.g., carboxylic acid,sulfonic acid).

In certain preferred embodiments, when the recognition moiety is acarboxylic acid, the recognition moiety will interact with the analyteby complexation (e.g., metal ions). In still other preferredembodiments, the carboxylic acid will protonate a basic group on theanalyte (e.g. amine).

In another preferred embodiment, the recognition moiety is a drugmoiety. The drug moieties can be agents already accepted for clinicaluse or they can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. The drug moieties can have aproven action in a given disease state or can be only hypothesized toshow desirable action in a given disease state. In a preferredembodiment, the drug moieties are compounds that are being screened fortheir ability to interact with an analyte of choice. As such, drugmoieties that are useful in practicing the instant invention includedrugs from a broad range of drug classes having a variety ofpharmacological activities.

Classes of useful agents include, for example, non-steroidalanti-inflammatory drugs (NSAIDS). The MAIDS can, for example, beselected from the following categories: (e.g., propionic acidderivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams); steroidalanti-inflammatory drugs including hydrocortisone and the like;antihistaminic drugs (e.g., chlorpheniranune, triprolidine); antitussivedrugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane);antipruritic drugs (e.g., methidilizine and trimeprizine);anticholinergic drugs (e.g., scopolamine, atropine, homatropine,levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine,meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); antiarrhythmic drugs (e.g., propanolol,procainamide, disopyraminde, quinidine, encamide); P-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, aminone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone,isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chlorprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer andsedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide,flurazapam, hydroxyzine, loxapine and promazine); antipsychotic drugs(e.g., chlorprothixene, fluphenazine, haloperidol, molindone,thioridazine and trifluoperazine); antimicrobial drugs (antibacterial,antifungal, antiprotozoal and antiviral drugs).

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, triclosan, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isothionate, metronidazole;pentamidine, gentamycin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmycin, paromomycin,streptomycin, tobramycin, miconazole, and amanfadine.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, a-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine).

The recognition moiety can also comprise hormones (e.g.,medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide orsomatostatin); muscle relaxant drugs (e.g., cinnamedrine,cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine,idaverine, ritodrine, dephenoxylate, dantrolene and azumolen);antispasmodic drugs; bone-active drugs (e.g., diphosphonate andphosphonoalkylphosphinate drug compounds); endocrine modulating drugs(e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol,norethindrone, mestranol, desogestrel, medroxyprogesterone), modulatorsof diabetes (e.g., glyburide or chlorpropamide), anabolics, such astestolactone or stanozolol, androgens (e.g., methyltestosterone,testosterone or fluoxymesterone), antidiuretics (e.g., desmopressin) andcalcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progenstogens, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful recognition moieties include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H₂ antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

When the recognition moiety is a chelating agent, crown ether orcyclodextrin, host-guest chemistry will dominate the interaction betweenthe recognition moiety and the analyte. The use of host-guest chemistryallows a great degree of recognition-moiety-analyte specificity to beengineered into a device of the invention. The use of these compounds tobind to specific compounds is well known to those of skill in the art.See, for example, Pitt et al. “The Design of Chelating Agents for theTreatment of Iron Overload,” In, INORGANIC CHEMISTRY IN BIOLOGY ANDMEDICINE; Martell, A. E., Ed.; American Chemical Society, Washington,D.C., 1980, pp. 279-312; Lindoy, L. F., THE CHEMISTRY OF MACROCYCLICLIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989; Dugas,H., BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, andreferences contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, R. E., Whitaker, I. R., Eds., American ChemicalSociety, Washington, D.C., 1982, pp. 370-387; Kasina et al. BioconjugateChem. 9:108-117 (1998); Song et al., Bioconjugate Chem. 8:249-255(1997).

In a presently preferred embodiment, the recognition moiety is apolyaminocarboxylate chelating agent such as ethylenediaminetetraaceticacid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). Theserecognition moieties can be attached to any amine-terminated componentof a SAM or a spacer arm, for example, by utilizing the commerciallyavailable dianhydride (Aldrich Chemical Co., Milwaukee, Wis.).

In still further preferred embodiments, the recognition moiety is abiomolecule such as a protein, nucleic acid, peptide or an antibody.Biomolecules useful in practicing the present invention can be derivedfrom any source. The biomolecules can be isolated from natural sourcesor can be produced by synthetic methods. Proteins can be naturalproteins or mutated proteins. Mutations can be effected by chemicalmutagenesis, site-directed mutagenesis or other means of inducingmutations known to those of skill in the art. Proteins useful inpracticing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal. Peptides and nucleic acids can be isolated from naturalsources or can be wholly or partially synthetic in origin.

In those embodiments wherein the recognition moiety is a protein orantibody, the protein can be tethered to a SAM component or a spacer armby any reactive peptide residue available on the surface of the protein.In preferred embodiments, the reactive groups are amines orcarboxylates. In particularly preferred embodiments, the reactive groupsare the e-amine groups of lysine residues. Furthermore, these moleculescan be adsorbed onto the surface of the substrate or SAM by non-specificinteractions (e.g., chemisorption, physisorption).

Recognition moieties that are antibodies can be used to recognizeanalytes which are proteins, peptides, nucleic acids, saccharides orsmall molecules such as drugs, herbicides, pesticides, industrialchemicals and agents of war. Methods of raising antibodies for specificmolecules are well-known to those of skill in the art. See, U.S. Pat.Nos. 5,147,786; 5,334,528; 5,686,237; 5,573,922; each of which isincorporated herein by reference. Methods for attaching antibodies tosurfaces are also art-known (See, Delamarche et al. Langmuir12:1944-1946 (1996)).

Peptides and nucleic acids can be attached to a SAM component or spacerarm. Both naturally-derived and synthetic peptides and nucleic acids areof use in conjunction with the present invention. These molecules can beattached to a SAM component or spacer arm by any available reactivegroup. For example, peptides can be attached through an amine, carboxyl,sulfhydryl, or hydroxyl group. Such a group can reside at a peptideterminus or at a site internal to the peptide chain. Nucleic acids canbe attached through a reactive group on a base (e.g., exocyclic amine)or an available hydroxyl group on a sugar moiety (e.g., 3′- or5′-hydroxyl). The peptide and nucleic acid chains can be furtherderivatized at one or more sites to allow for the attachment ofappropriate reactive groups onto the chain (See, Chrisey et al. NucleicAcids Res. 24:3031-3039 (1996)).

When the peptide or nucleic acid is a fully or partially syntheticmolecule, a reactive group or masked reactive group can be incorporatedduring the process of the synthesis. Many derivatized monomersappropriate for reactive group incorporation in both peptides andnucleic acids are know to those of skill in the art (See, for example,THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY, Vol. 2: “Special Methods inPeptide Synthesis,” Gross, E. and Melenhofer, J., Eds., Academic Press,New York (1980)). Many useful monomers are commercially available(Bachem, Sigma, etc.). This masked group can then be unmasked followingthe synthesis, at which time it becomes available for reaction with aSAM component or a spacer arm.

In other preferred embodiments, the peptide is attached directly to thesubstrate (See, Frey et al. Anal. Chem. 68:3187-3193 (1996)). In aparticularly preferred embodiment, the peptide is attached to a goldsubstrate through a sulfhydryl group on a cysteine residue. In anotherpreferred embodiment, the peptide is attached through a thiol to aspacer arm which terminates in, for example, an iodoacetamide,chloroacetamide, benzyl iodide, benzyl bromide, alkyl iodide or alkylbromide. Similar immobilization techniques are known to those of skillin the art (See, for example, Zull et al. J. Ind Microbiol. 13:137-143(1994)).

In another preferred embodiment, the recognition moiety forms aninclusion complex with the analyte of interest. In a particularlypreferred embodiment, the recognition moiety is a cyclodextrin ormodified cyclodextrin. Cyclodextrins are a group of cyclicoligosaccharides produced by numerous microorganisms. Cyclodextrins havea ring structure which has a basket-like shape. This shape allowscyclodextrins to include many kinds of molecules into their internalcavity (See, for example, Szejtli, J., CYCLODEXTRINS AND THEIR INCLUSIONCOMPLEXES; Akademiai Klado, Budapest, 1982; and Bender et al.,CYCLODEXTRIN CHEMISTRY, Springer-Verlag, Berlin, 1978).

Cyclodextrins are able to form inclusion complexes with an array oforganic molecules including, for example, drugs, pesticides, herbicidesand agents of war (See, Tenjarla et al., J. Pharm. Sci. 87:425-429(1998); Zughul et al., Pharm. Dev. Technol. 3:43-53 (1998); and Alberset al., Crit. Rev. Ther. Drug Carrier Syst. 12:311-337 (1995)).Importantly, cyclodextrins are able to discriminate between enantiomersof compounds in their inclusion complexes. Thus, in one preferredembodiment, the invention provides for the detection of a particularenantiomer in a mixture of enantiomers (See, Koppenhoefer et al. J.Chromatogr. A 793:153-164 (1998)).

The cyclodextrin recognition moiety can be attached to a SAM component,through a spacer arm or directly to the substrate (See, Yamamoto et al.,J. Phys. Chem. B 101:6855-6860 (1997)). Methods to attach cyclodextrinsto other molecules are well known to those of skill in thechromatographic and pharmaceutical arts (See, Sreenivasan, Appl. Polym.Sci. 60:2245-2249 (1996)).

In other embodiments, the recognition moieties can be nucleic acids(e.g., RNA or DNA) or receptors that are specific for a particularentity (e.g., virus). In some embodiments, the nucleic acids areaptamers. The isolation of aptamers is described in U.S. Pat. Nos.5,475,096; 5,270,163; and 5,475,096; and in PCT publications WO97/38134, WO 98/33941, and WO 99/07724, all of which are hereinincorporated by reference.

In some embodiments, recognition moieties are incorporated to detect avariety of bacteria and pathogens. Such recognition moieties include,but not limited to, sialic acid to detect HIV (Wies et al., Nature 333:426 [1988]), influenza (White et al., Cell 56: 725 [1989]), chlamydia(Infect. 1 mm. 57: 2378 [1989]), reovirus, Streptococcus suis,Salmonella, Sendai virus, mumps, newcastle, myxovirus, and Neisseriameningitidis; 9-OAC sialic acid to detect coronavirus, encephalomyelitisvirus, and rotavirus; non-sialic acid glycoproteins to detectcytomegalovirus (Virology 176: 337 [1990]) and measles virus (Virology172: 386 [1989]); CD4 (Khatzman et al., Nature 312: 763 [1985]),vasoactive intestinal peptide (Sacerdote et al., J. of NeuroscienceResearch 18: 102 [1987]), and peptide T (Ruff et al., FEBS Letters 211:17 [1987]) to detect HIV; epidermal growth factor to detect vaccinia(Epstein et al., Nature 318: 663 [1985]); acetylcholine receptor todetect rabies (Lentz et al., Science 215: 182 [1982]); Cd3 complementreceptor to detect Epstein-Barr virus (Carel et al., J. Biol. Chem. 265:12293 [1990]); β-adrenergic receptor to detect rheovirus (Co et al.,Proc. Natl. Acad. Sci. 82: 1494 [1985]); ICAM-1 (Marlin et al., Nature344: 70 [1990]), N-CAM, and myelin-associated glycoprotein MAb (Shepheyet al., Proc. Natl. Acad. Sci. 85: 7743 [1988]) to detect rhinovirus;polio virus receptor to detect polio virus (Mendelsohn et al., Cell 56:855 [1989]); fibroblast growth factor receptor to detect herpesvirus(Kaner et al., Science 248: 1410 [1990]); oligomannose to detectEscherichia coli; ganglioside GM1 to detect Neisseria meningitidis; andantibodies to detect a broad variety of pathogens (e.g., Neisseriagonorrhoeae, V. vulnificus, V. parahaemolyticus, V. cholerae, V.alginolyticus, etc.).

In still further embodiments, the recognition moiety is a ligand thatinteracts with a binding partner. Examples of ligands include, but arenot limited to, small organic molecules such as steroid molecules andsmall drug molecules, proteins, polypeptides and peptides, metal ions,and nucleic acids. In some embodiments, the ligand is recognized by abinding molecule in a sample. Examples of binding molecules include, butare not limited to, steroids, hormones, proteins, polypeptides, andpeptides such immunoglobulin molecules and fragments thereof, nucleicacids, and other organic or non-organic molecules. In some preferredembodiments, the ligand is recognized by a binding molecule in a bodyfluid of a test subject. For example, the ligand can be a virus envelopeprotein or some other antigenic molecule from a pathogenic organism(such as those listed above). In preferred embodiments, the antigenicmolecule (e.g., a protein) is recognized by an antibody molecule in thebody fluid of a test subject that has been exposed to the pathogenicorganism. In particularly preferred embodiments, the ligand is protein Efrom the envelope of West Nile Virus.

In some preferred embodiments, the ligands or recognition moieties arecomplexed with a lipid. The present invention contemplates complexationof the recognition moiety with a variety of lipids and lipid containingmaterials, including, but not limited to, fatty acids, phospholipids,mono-, di- and tri-glycerides comprising fatty acids and/orphospholipids, lipid bilayers, and liposomes. The lipid containingmaterial can be provided as multilayers, as well as braided, lamellar,helical, tubular, and fiber-like shapes, and combinations thereof.Standard attachment chemistries are available for attaching arecognition moiety or ligand of interest to lipids and lipids containingmaterials. These attachment chemistries are described in more detailbelow with reference to liposomes.

In some preferred embodiments, the present invention utilizes liposomes.A variety of methods are useful for producing liposomes. Such methodsare described in detail in numerous articles and have been reviewed intexts such as New (New, Liposomes: A Practical Approach, IRL Press,Oxford, [1989]), and Rosoff (Rosoff, Vesicles, Marcel Dekker, Inc., NewYork, [1996]) among others. See also, U.S. Pat. Nos. 6,183,772,6,306,598, 6,180,784, 6,740,643, and 6,706,922, all of which areincorporated herein by reference, for methods of forming liposomes andother lipid containing materials. In some preferred embodiments, theliposomes are prepared using a probe sonication methods. Methods ofderivatizing lipids with a diverse range of compounds (e.g.,carbohydrates, proteins, nucleic acids, and other chemical groups) arewell known in the art. The carboxylic acid on the terminal end of lipidscan be easily modified to form esters, phosphate esters, amino groups,ammoniums, hydrazines, polyethylene oxides, amides, and many othercompounds. These chemical groups provide linking groups forcarbohydrates, proteins, nucleic acids, and other chemical groups (e.g.,carboxylic acids can be directly linked to proteins by making theactivated ester, followed by reaction to free amine groups on a proteinto form an amide linkage). Examples of antibodies attached to Langmuirfilms are known in the art (See e.g., Tronin et al., Langmuir 11: 385[1995]; and Vikhohn et al., Langmuir 12: 3276 [1996]). There arenumerous other means to couple materials to membranes, or incorporatematerials within a membrane, including for example, coupling of proteinsor nucleic acids to polymer membranes (See e.g., Bamford et al. Adv.Mat. 6: 550 [1994]); coupling of proteins to self-assembled organicmonolayers (See e.g., Willner et al., Adv. Mat. 5: 912 [1993]), andincorporating proteins into membranes (See e.g., Downer et al.,Biosensor and Bioelect. 7: 429 [1992]); among others. Ligands (e.g.proteins, nucleic acids, and carbohydrates) can be conveniently attachedto the derivatized lipids.

In some embodiments, ligands or recognition moieties are covalentlylinked to the head groups of lipid monomers. In other embodiments,ligands or recognition moieties are covalently linked to the surface ofa lipid containing material (e.g., proteins and antibodies with multipleamine and thiol linkages to the material surface). Ins till otherembodiments, ligands or recognition moieties are non-covalentlyincorporated into the biopolymeric material (e.g., gangliosideincorporated into the membrane of films and liposomes).

II. Substrates

Substrates that are useful in practicing the present invention can bemade of practically any physicochemically stable material. In apreferred embodiment, the substrate material is non-reactive towards theconstituents of the mesogenic layer. The substrates can be either rigidor flexible and can be either optically transparent or optically opaque.The substrates can be electrical insulators, conductors orsemiconductors. Further, the substrates can be substantially impermeableto liquids, vapors and/or gases or, alternatively, the substrates can bepermeable to one or more of these classes of materials. Exemplarysubstrate materials include, but are not limited to, inorganic crystals,inorganic glasses, inorganic oxides, metals, organic polymers andcombinations thereof. In some embodiments, the substrates havemicrochannels therein for the delivery of sample and/or other reagentsto the substrate surface or detection regions thereon. The design anduse of microchannels are described, for example, in U.S. Pat. Nos.6,425,972, 6,418,968, 6,447,727, 6,432,720, 5,976,336, 5,882,465,5,876,675, 6,186,660, 6,100,541, 6,379,974, 6,267,858, 6,251,343,6,238,538, 6,182,733, 6,068,752, 6,429,025, 6,413,782, 6,274,089,6,150,180, 6,046,056, 6,358,387, 6,321,791, 6,326,083, 6,171,067, and6167910, all of which are incorporated herein by reference.

A. Inorganic Crystal and Glasses

In some embodiments of the present invention, inorganic crystals andinorganic glasses are utilized as substrate materials (e.g., LiF, NaF,NaCl, KBr, KI, CaF₂, MgF₂, HgF₂, BN, AsS₃, ZnS, Si₃N₄ and the like). Thecrystals and glasses can be prepared by art standard techniques (See,e.g., Goodman, C. H. L., Crystal Growth Theory and Techniques, PlenumPress, New York 1974). Alternatively, the crystals can be purchasedcommercially (e.g., Fischer Scientific). The crystals can be the solecomponent of the substrate or they can be coated with one or moreadditional substrate components. Thus, it is within the scope of thepresent invention to utilize crystals coated with, for example one ormore metal films or a metal film and an organic polymer. Additionally, acrystal can constitute a portion of a substrate which contacts anotherportion of the substrate made of a different material, or a differentphysical form (e.g., a glass) of the same material. Other usefulsubstrate configurations utilizing inorganic crystals and/or glasseswill be apparent to those of skill in the art.

B. Inorganic Oxides

In other embodiments of the present invention, inorganic oxides areutilized as the substrate. Inorganic oxides of use in the presentinvention include, for example, Cs₂0, Mg(OH)₂, Ti0₂, Zr0₂, Ce0₂, Y₂0₃,Cr₂0₃, Fe₂0₃, NiO, ZnO, Al₂0₃, Si0₂ (glass), quartz, In₂0₃, Sn0₂, Pb0₂and the like. The inorganic oxides can be utilized in a variety ofphysical forms such as films, supported powders, glasses, crystals andthe like. A substrate can consist of a single inorganic oxide or acomposite of more than one inorganic oxide. For example, a composite ofinorganic oxides can have a layered structure (i.e., a second oxidedeposited on a first oxide) or two or more oxides can be arranged in acontiguous non-layered structure. In addition, one or more oxides can beadmixed as particles of various sizes and deposited on a support such asa glass or metal sheet. Further, a layer of one or more inorganic oxidescan be intercalated between two other substrate layers (e.g.,metal-oxide-metal, metal-oxide-crystal).

In a presently preferred embodiment, the substrate is a rigid structurethat is impermeable to liquids and gases. In this embodiment, thesubstrate consists of a glass plate onto which a metal, such as gold islayered by evaporative deposition. In a still further preferredembodiment, the substrate is a glass plate (Si0₂) onto which a firstmetal layer such as titanium has been layered. A layer of a second metalsuch as gold is then layered on top of the first metal layer.

C. Metals

In still further embodiments of the present invention, metals areutilized as substrates. The metal can be used as a crystal, a sheet or apowder. The metal can be deposited onto a backing by any method known tothose of skill in the art including, but not limited to, evaporativedeposition, sputtering, electroless deposition, electrolytic depositionand adsorption or deposition of preform particles of the metal includingmetallic nanoparticles.

Any metal that is chemically inert towards the mesogenic layer will beuseful as a substrate in the present invention. Metals that are reactiveor interactive towards the mesogenic layer will also be useful in thepresent invention. Metals that are presently preferred as substratesinclude, but are not limited to, gold, silver, platinum, palladium,nickel and copper. In one embodiment, more than one metal is used. Themore than one metal can be present as an alloy or they can be formedinto a layered “sandwich” structure, or they can be laterally adjacentto one another. In a preferred embodiment, the metal used for thesubstrate is gold. In a particularly preferred embodiment the metal usedis gold layered on titanium.

The metal layers can be either permeable or impermeable to materialssuch as liquids, solutions, vapors and gases.

D. Organic Polymers

In still other embodiments of the present invention, organic polymersare utilized as substrate materials. Organic polymers useful assubstrates in the present invention include polymers that are permeableto gases, liquids and molecules in solution. Other useful polymers arethose that are impermeable to one or more of these same classes ofcompounds.

Organic polymers that form useful substrates include, for example,polyalkenes (e.g., polyethylene, polyisobutene, polybutadiene),polyacrylics (e.g., polyacrylate, polymethyl methacrylate,polycyanoacrylate), polyvinyls (e.g., polyvinyl alcohol, polyvinylacetate, polyvinyl butyral, polyvinyl chloride), polystyrenes,polycarbonates, polyesters, polyurethanes, polyamides, polyimides,polysulfone, polysiloxanes, polyheterocycles, cellulose derivative(e.g., methyl cellulose, cellulose acetate, nitrocellulose),polysilanes, fluorinated polymers, epoxies, polyethers and phenolicresins (See, Cognard, J. ALIGNMENT OF NEMATIC LIQUID CRYSTALS AND THEIRMIXTURES, in Mol. Cryst. Liq. Cryst. 1:1-74 (1982)). Presently preferredorganic polymers include polydimethylsiloxane, polyethylene,polyacrylonitrile, cellulosic materials, polycarbonates and polyvinylpyridinium.

In a presently preferred embodiment, the substrate is permeable and itconsists of a layer of gold, or gold over titanium, which is depositedon a polymeric membrane, or other material, that is permeable toliquids, vapors and/or gases. The liquids and gases can be purecompounds (e.g., chloroform, carbon monoxide) or they can be compoundswhich are dispersed in other molecules (e.g., aqueous protein solutions,herbicides in air, alcoholic solutions of small organic molecules).Useful permeable membranes include, but are not limited to, flexiblecellulosic materials (e.g., regenerated cellulose dialysis membranes),rigid cellulosic materials (e.g., cellulose ester dialysis membranes),rigid polyvinylidene fluoride membranes, polydimethylsiloxane and tracketched polycarbonate membranes.

In a further preferred embodiment, the layer of gold on the permeablemembrane is itself permeable. In a still further preferred embodiment,the permeable gold layer has a thickness of about 70 Angstroms or less.

In those embodiments wherein the permeability of the substrate is not aconcern and a layer of a metal film is used, the film can be as thick asis necessary for a particular application. For example, if the film isused as an electrode, the film can be thicker than in an embodiment inwhich it is necessary for the film to be transparent or semi-transparentto light.

Thus, in a preferred embodiment, the film is of a thickness of fromabout 0.01 nanometer to about 1 micrometer. In a further preferredembodiment, the film is of a thickness of from about 5 nanometers toabout 100 nanometers. In yet a further preferred embodiment, the film isof a thickness of from about 10 nanometers to about 50 nanometers.

E. Formats

The substrates of the present invention are provided in a variety offormats. For examples, the substrates may present planar or curvedsurfaces or be beads. The bead format is especially useful for theindirect detection methods described below. The bead substrates of thepresent invention may comprise any of the substrate materials describedabove. In some preferred embodiments, the beads are commerciallyavailable beads such as agarose beads, acrylic beads, or latex beads. Insome embodiments, the beads are magnetic. In still other embodiments,the beads are coated with a metal such as silver or gold. In still otherembodiments, substrates such column chromatography media may be used tocapture analytes. Examples of such substrates include immunoaffinitycolumns (i.e., columns containing media functionalized with antigenbinding proteins), protein-A affinity columns, cation exchange columnssuch as S-SEPHAROSE, SP-SEPHAROSE, and carboxymethyl cellulose, anionexchange columns such as DEAE Cellulose, QAE SEPHADEX, and FAST QSEPHAROSE, sizing columns such as ULTRAGEL columns, phsosphocellusecolumns, heparin sulfate columns, and the like. Following elution forthe columns analytes are detected as described in detail below.

III. Functionalization of Substrates

In some embodiments, the surface of the substrate is functionalized sothat a recognition moiety is immobilized on the surface of thesubstrate. In some embodiments, the immobilized recognition moiety formsa detection region. In some embodiments, a plurality of detectionregions are formed on the surface of the substrate. In some embodiments,the same recognition moiety is provided on two or more of the pluralityof detection regions, while in other embodiments, at least two differentrecognition moieties are immobilized on one or more of the plurality ofdetection regions. In some embodiments, the recognition moieties arearrayed in discreet detection regions on the substrate surfaces by themethods described in more detail below.

A. Self-Assembled Monolayers

In some embodiments, the surface of the substrate is firstfunctionalized by forming a self-assembled monolayer (SAM) on thesubstrate surface. Self-assembled monolayers are generally depicted asan assembly of organized, closely packed linear molecules. There are twowidely-used methods to deposit molecular monolayers on solid substrates:Langmuir-Blodgett transfer and self-assembly. Additional methods includetechniques such as depositing a vapor of the monolayer precursor onto asubstrate surface and the layer-by-layer deposition of polymers andpolyelectrolytes from solution (Ladam et al., Protein Adsorption ontoAuto-Assembled Polyelectrolyte Films, Langmuir; 2001; 17(3); 878-882).

The composition of a layer of a SAM useful in the present invention canbe varied over a wide range of compound structures and molar ratios. Inone embodiment, the SAM is formed from only one compound. In a presentlypreferred embodiment, the SAM is formed from two or more components. Inanother preferred embodiment, when two or more components are used, onecomponent is a long-chain hydrocarbon having a chain length of between10 and 25 carbons and a second component is a short-chain hydrocarbonhaving a chain length of between 1 and 9 carbon atoms. In particularlypreferred embodiments, the SAM is formed from CH₃(CH₂)₁₅SH andCH₃(CH₂)₄SH or CH₃(CH₂)₁₅SH and CH₃(CH₂)₉SH. In any of the abovedescribed embodiments, the carbon chains can be functionalized at theco-terminus (e.g., NH₂, COOH, OH, CN), at internal positions of thechain (e.g., aza, oxa, thia) or at both the co-terminus and internalpositions of the chain.

A recognition moiety can be attached to the surface of a SAM by any of alarge number of art-known attachment methods. In one preferredembodiment, a reactive SAM component is attached to the substrate andthe recognition moiety is subsequently bound to the SAM component viathe reactive group on the component and a group of complementaryreactivity on the recognition moiety (See, e.g., Hegner et al. Biophys.J. 70:2052-2066 (1996)). In another preferred embodiment, therecognition moiety is attached to the SAM component prior toimmobilizing the SAM component on the substrate surface: the recognitionmoiety-SAM component cassette is then attached to the substrate. In astill further preferred embodiment, the recognition moiety is attachedto the substrate via a displacement reaction. In this embodiment, theSAM is preformed and then a fraction of the SAM components are displacedby a recognition moiety or a SAM component bearing a virus recognitionmoiety. In still other embodiments, the polypeptide recognition moietiesare adsorbed directly onto hydrophobic monolayers such as CH₃(CH₂)₁₅SH.In embodiments where the recognition moiety is an antibody or othermolecule that binds to protein A, protein A is first attached to themonolayer followed by the antibody, which is bound by protein A.

B. Functionalized SAMs

The discussion which follows focuses on the attachment of a reactive SAMcomponent to the substrate surface. This focus is for convenience onlyand one of skill in the art will understand that the discussion isequally applicable to embodiments in which the SAM component-recognitionmoiety is preformed prior to its attachment to the substrate. As usedherein, “reactive SAM components” refers to components that have afunctional group available for reaction with a recognition moiety orother species following the attachment of the component to thesubstrate.

Currently favored classes of reactions available with reactive SAMcomponents are those that proceed under relatively mild conditions.These include, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides), electrophilicsubstitutions (e.g., enamine reactions) and additions to carbon-carbonand carbon-heteroatom multiple bonds (e.g., Michael reaction,Diels-Alder addition). These and other useful reactions are discussed inMarch, ADVANCED ORGANIC CHEMISTRY, Third Ed., John Wiley & Sons, NewYork, 1985.

According to the present invention, a substrate's surface isfunctionalized with SAM, components and other species by covalentlybinding a reactive SAM component to the substrate surface in such a wayas to derivatize the substrate surface with a plurality of availablereactive functional groups. Reactive groups which can be used inpracticing the present invention include, for example, amines, hydroxylgroups, carboxylic acids, carboxylic acid derivatives, alkenes,sulfhydryls, siloxanes, etc.

A wide variety of reaction types are available for the functionalizationof a substrate surface. For example, substrates constructed of a plasticsuch as polypropylene, can be surface derivatized by chromic acidoxidation, and subsequently converted to hydroxylated or aminomethylatedsurfaces. Substrates made from highly crosslinked divinylbenzene can besurface derivatized by chloromethylation and subsequent functional groupmanipulation. Additionally, functionalized substrates can be made frometched, reduced polytetrafluoroethylene.

When the substrates are constructed of a siliaceous material such asglass, the surface can be derivatized by reacting the surface Si—OH,Si0-H, and/or Si—Si groups with a functionalizing reagent. When thesubstrate is made of a metal film, the surface can be derivatized with amaterial displaying avidity for that metal.

In a preferred embodiment, wherein the substrates are made from glass,the covalent bonding of the reactive group to the glass surface isachieved by conversion of groups on the substrate's surface by a siliconmodifying reagent such as:

(RO)₃—Si—R¹—X¹  (1)

where R is an alkyl group, such as methyl or ethyl, R¹ is a linkinggroup between silicon and X and X is a reactive group or a protectedreactive group. The reactive group can also be a recognition moiety asdiscussed below. Silane derivatives having halogens or other leavinggroups beside the displayed alkoxy groups are also useful in the presentinvention.

A number of siloxane functionalizing reagents can be used, for example:

-   -   1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with        diborane, and H₂0₂ to oxidize the alcohol)        -   a. allyl trichlorosilane→→3-hydroxypropyl        -   b. 7-oct-1-enyl trichlorosilane→→8-hydroxyoctyl    -   2. Diol (dihydroxyalkyl) siloxanes (silylate surface and        hydrolyze to diol)        -   a. (glycidyl            trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl    -   3. Aminoalkyl siloxanes (amines requiring no intermediate        functionalizing step).        -   a. 3-aminopropyl trimethoxysilane→aminopropyl    -   4. Dimeric secondary aminoalkyl siloxanes        -   a. bis(3-trimethoxysilylpropyl)            amine→bis(silyloxylpropyl)amine.

It will be apparent to those of skill in the art that an array ofsimilarly useful functionalizing chemistries are available when SAMcomponents other than siloxanes are used. Thus, for example similarlyfunctionalized alkyl thiols can be attached to metal films andsubsequently reacted to produce the functional groups such as thoseexemplified above.

In another preferred embodiment, the substrate is at least partially ametal film, such as a gold film, and the reactive group is tethered tothe metal surface by an agent displaying avidity for that surface. In apresently preferred embodiment, the substrate is at least partially agold film and the group which reacts with the metal surface comprises athiol, sulfide or disulfide such as:

Y—S—R²—X²  (2)

R² is a linking group between sulfur and X² and X² is a reactive groupor a protected reactive group. X² can also be a recognition moiety asdiscussed below. Y is a member selected from the group consisting of H,R³ and R³—S—, wherein R² and R³ are independently selected. When R² andR³ are the same, symmetrical sulfides and disulfides result, and whenthey are different, asymmetrical sulfides and disulfides result.

A large number of functionalized thiols, sulfides and disulfides arecommercially available (Aldrich Chemical Co., St. Louis). Additionally,those of skill in the art have available to them a manifold of syntheticroutes with which to produce additional such molecules. For example,amine-functionalized thiols can be produced from the correspondinghalo-amines, halo-carboxylic acids, etc. by reaction of these haloprecursors with sodium sulfhydride. See, e.g., Reid, ORGANIC CHEMISTRYof BIVALENT SULFUR, VOL 1, pp. 21-29, 32-35, vol. 5, pp. 27-34, ChemicalPublishing Co., New York, 1.958, 1963. Additionally, functionalizedsulfides can be prepared via alkylthio-de-halogenation with a mercaptansalt (See, Reid, ORGANIC CHEMISTRY OF BIVALENT SULFUR, vol. 2, pp.16-21, 24-29, vol. 3, pp. 11-14, Chemical Publishing Co., New York,1960). Other methods for producing compounds useful in practicing thepresent invention will be apparent to those of skill in the art.

In another preferred embodiment, the functionalizing reagent providesfor more than one reactive group per each reagent molecule. Usingreagents such as Compound 3, below, each reactive site on the substratesurface is, in essence, “amplified” to two or more functional groups:

(RO)₃—Si—R²—(X²)_(n)  (3)

where R is an alkyl group, such as methyl, R² is a linking group betweensilicon and X², X² is a reactive group or a protected reactive group andn is an integer between 2 and 50, and more preferably between 2 and 20.

Similar amplifying molecules are also of use in those embodimentswherein the substrate is at least partially a metal film. In theseembodiments the group which reacts with the metal surface comprises athiol, sulfide or disulfide such as in Formula (4):

Y—S—R²—(X²)_(n)  (4)

As discussed above, R² is a linking group between sulfur and X² and X²is a reactive group or a protected reactive group. X² can also be arecognition moiety. Y is a member selected from the group consisting ofH, R³ and R³—S—, wherein R² and R³ are independently selected.

R groups of use for R¹, R² and R³ in the above described embodiments ofthe present invention include, but are not limited to, alkyl,substituted alkyl, aryl, arylalkyl, substituted aryl, substitutedarylalkyl, acyl, halogen, hydroxy, amino, alkylamino, acylamino, alkoxy,acyloxy, aryloxy, aryloxyalkyl, mercapto, saturated cyclic hydrocarbon,unsaturated cyclic hydrocarbon, heteroaryl, heteroarylalkyl, substitutedheteroaryl, substituted heteroarylalkyl, heterocyclic, substitutedheterocyclic and heterocyclicalkyl groups.

In each of Formulae 1-4, above, each of R¹, R² and R³ are either stableor they can be cleaved by chemical or photochemical reactions. Forexample, R groups comprising ester or disulfide bonds can be cleaved byhydrolysis and reduction, respectively. Also within the scope of thepresent invention is the use of R groups which are cleaved by light suchas, for example, nitrobenzyl derivatives, phenacyl groups, benzoinesters, etc. Other such cleaveable groups are well-known to those ofskill in the art.

In another preferred embodiment, the organosulfur compound is partiallyor entirely halogenated. An example of compounds useful in thisembodiment include:

X¹Q₂C(CQ¹ ₂)_(m)Z¹(CQ² ₂)_(n)SH  (5)

wherein, X¹ is a member selected from the group consisting of H, halogenreactive groups and protected reactive groups. Reactive groups can alsobe recognition moieties as discussed below. Q, Q¹ and Q² areindependently members selected from the group consisting of H andhalogen. Z¹ is a member selected from the group consisting of —CQ₂-,—CQ¹ ₂—, —CQ² ₂—, —O—, —S—, NR⁴—, —C(O)NR⁴ and R⁴NC(O0-, in which R⁴ isa member selected from the group consisting of H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups and mand n are independently a number between 0 and 40.

In yet another preferred embodiment, the organic layer comprises acompound according to Formula 5 above, in which Q, Q¹ and Q² areindependently members selected from the group consisting of H andfluorine. In a still further preferred embodiment, the organic layercomprises compounds having a structure according to Formulae (6) and(7):

CF₃(CF₂)_(m)Z¹(CH₂)_(n)SH  (6)

CF₃(CF₂)_(o)Z²(CH₂)_(p)SH  (7)

wherein, Z¹ and Z² are members independently selected from the groupconsisting of —CH₂—, —O—, —S—, NR⁴, —C(O)NR⁴ and R⁴NC(O)— in which R⁴ isa member selected from the group consisting of H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups. In apresently preferred embodiment, the Z groups of adjacent moleculesparticipate in either an attractive (e.g., hydrogen bonding) orrepulsive (e.g., van der Waals) interaction.

In Formula 7, m is a number between 0 and 40, n is a number between 0and 40, o is a number between 0 and 40 and p is a number between 0 and40.

In a further preferred embodiment, the compounds of Formulae 6 and 7 areused in conjunction with an organosulfur compound, either halogentatedor unhalogenated, that bears a recognition moiety.

When the organic layer is formed from a halogenated organosulfurcompound, the organic layer can comprise a single halogenated compoundor more than one halogenated compound having different structures.Additionally, these layers can comprise a non-halogenated organosulfurcompound.

The reactive functional groups (X¹ and X²) are, for example:

(a) carboxyl groups and various derivatives thereof including, but notlimited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.

(c) haloalkyl groups wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups which can be converted to disulfides or reacted withacyl halides;

(h) amine or sulfhydryl groups which can be, for example, acylated oralkylated;

(i) alkenes which can undergo, for example, cycloadditions, acylation,Michael addition, etc; and

(j) epoxides which can react with, for example, amines and hydroxylcompounds.

The reactive moieties can also be recognition moieties. The nature ofthese groups is discussed in greater detail below.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reaction controlling theattachment of the functionalized SAM component onto the substrate'ssurface. Alternatively, the reactive functional group can be protectedfrom participating in the reaction by the presence of a protectinggroup. Those of skill in the art will understand how to protect aparticular functional group from interfering with a chosen set ofreaction conditions. For examples of useful protecting groups, seeGreene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley &Sons, New York, 1991.

In a preferred embodiment, the SAM component bearing the recognitionmoiety is attached directly and essentially irreversibly via a “stablebond” to the surface of the substrate. A “stable bond”, as used herein,is a bond which maintains its chemical integrity over a wide range ofconditions (e.g., amide, carbamate, carbon-carbon, ether, etc.). Inanother preferred embodiment, the SAM component bearing the recognitionmoiety is attached to the substrate surface by a “cleaveable bond”. A“cleaveable bond”, as used herein, is a bond that is designed to undergoscission under conditions which do not degrade other bonds in therecognition moiety-analyte complex. Cleaveable bonds include, but arenot limited to, disulfide, imine, carbonate and ester bonds.

In certain embodiments, it is advantageous to have the recognitionmoiety attached to a SAM component having a structure that is differentthan that of the constituents of the bulk SAM. In this embodiment, thegroup to which the recognition moiety is bound is referred to as a“spacer arm” or “spacer.” Using such spacer arms, the properties of theSAM adjacent to the recognition moiety can be controlled. Propertiesthat are usefully controlled include, for example, hydrophobicity,hydrophilicity, surface-activity and the distance of the recognitionmoiety from the plane of the substrate and/or the SAM. For example, in aSAM composed of alkanethiols, the recognition moiety can be attached tothe substrate or the surface of the SAM via an amine terminatedpoly(ethyleneglycol). Numerous other combinations of spacer arms andSAMs are accessible to those of skill in the art.

The hydrophilicity of the substrate surface can be enhanced by reactionwith polar molecules such as amine-, hydroxyl- andpolyhydroxylcontaining molecules. Representative examples include, butare not limited to, polylysine, polyethyleneimine, poly(ethyleneglycol)and poly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art (See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.1991).

The hydrophobicity of the substrate surface can be modulated by using ahydrophobic spacer arm such as, for example, long chain diamines, longchain thiols, α, ω-amino acids, etc. Representative hydrophobic spacersinclude, but are not limited to, 1,6-hexanediamine, 1,8-octanediamine,6-aminohexanoic acid and 8-aminooctanoic acid.

The substrate surface can also be made surface-active by attaching tothe substrate surface a spacer which has surfactant properties.Compounds useful for this purpose include, for example, aminated orhydroxylated detergent molecules such as, for example, 1-aminododecanoicacid.

In another embodiment, the spacer serves to distance the virusrecognition moiety from the substrate or SAM. Spacers with thischaracteristic have several uses. For example, a recognition moiety heldtoo closely to the substrate or SAM surface may not react with incominganalyte, or it may react unacceptably slowly. When an analyte is itselfsterically demanding, the reaction leading to recognition moiety-analytecomplex formation can be undesirably slowed, or not occur at all, due tothe monolithic substrate hindering the approach of the two components.

In another embodiment, the physicochemical characteristics (e.g.,hydrophobicity, hydrophilicity, surface activity, conformation) of thesubstrate surface and/or SAM are altered by attaching a monovalentmoiety which is different in composition than the constituents of thebulk SAM and which does not bear a recognition moiety. As used herein,“monovalent moiety” refers to organic molecules with only one reactivefunctional group. This functional group attaches the molecule to thesubstrate. “Monovalent moieties” are to be contrasted with thebifunctional “spacer” groups described above. Such monovalent groups areused to modify the hydrophilicity, hydrophobicity, bindingcharacteristics, etc. of the substrate surface. Examples of groupsuseful for this purpose include long chain alcohols, amines, fattyacids, fatty acid derivatives, poly(ethyleneglycol) monomethyl ethers,etc.

When two or more structurally distinct moieties are used as componentsof the SAMs, the components can be contacted with the substrate as amixture of SAM components or, alternatively, the components can be addedindividually. In those embodiments in which the SAM components are addedas a mixture, the mole ratio of a mixture of the components in solutionresults in the same ratio in the mixed SAM. Depending on the manner inwhich the SAM is assembled, the two components do not phase segregateinto islands (See, Bain and Whitesides, J. Am. Chem. Soc. 111:7164(1989)). This feature of SAMs can be used to immobilize recognitionmoieties or bulky modifying groups in such a manner that certaininteractions, such as steric hindrance, between these molecules isminimized.

The individual components of the SAMs can also be bound to the substratein a sequential manner. Thus, in one embodiment, a first SAM componentis attached to the substrate's surface by “underlabeling” the surfacefunctional groups with less than a stoichiometric equivalent of thefirst component. The first component can be a SAM component liked to aterminal reactive group or recognition group, a spacer arm or amonovalent moiety. Subsequently, the second component is contacted withthe substrate. This second component can either be added instoichiometric equivalence, stoichiometric excess or can again be usedto underlabel to leave sites open for a third component.

C. Polyimides

In some embodiments, the substrates are coated with polyimide layer. Itis contemplated that polyimide coated substrates are especially usefulbecause in some instances, the surfaces homeotropically orient a liquidcrystal, while in other instances the surfaces can be rubbed to providean anisotropic surface for orient a liquid crystal. In preferredembodiments, a substrate such as a silicon wafer is coated with apolyimide. In preferred embodiment, the substrate is spin coated withthe polyimide. A variety of polyimides find use with the presentinvention, including, but not limited to Nissan 7210, Nissan 3510,Nissan 410, Nissan 3140, Nissan 5291, and Japan Synthetic Rubber JALS146-R19 for planar alignment of liquid crystals and Nissan 7511L and SE1211 for homeotropic orientation of liquid crystals. Surprising, it hasbeen found that the ability of rubbed polyimide surfaces to orientliquid crystals is maintained when a recognition moiety is displayed onthe rubbed surface, and then masked when an analyte binds therecognition moiety. Thus, areas where an analyte is bound have anon-ordered liquid crystal and appear white or bright when viewedthrough cross polars and areas where analyte is not bound remain orderedand appear dark when viewed through cross polars. Surprising, it hasalso been found that polyimide surfaces that homeotropically orientliquid crystals can be used to report non-specific binding to thesurface. In these embodiments, areas where an analyte is bound have adisordered liquid crystal appear white or bright when viewed throughcross polars and areas where no analyte is bound maintain thehomeotropic orientation and appear dark. These different polyimidesprovide different anchoring properties and different binding affinity todifferent proteins which can be used to probe and report the bindingevents between the proteins. Likewise, different liquid crystals showdifferent response to the specific binding event. Therefore, it ispossible to tune the assays by using different liquid crystallinematerials such as, 5CB, BL093, TL 216, ZLI 5800, MLC 6613, and(p-methoxybenzylidene)-p-butylaniline (MBBA) with different optical anddielectric properties.

D. Direct Adsorption

In some embodiments, the recognition moiety is immobilized on asubstrate by direct adsorption. For example, an antibody can beimmobilized onto a thin film of polyurethane spin coated onto a goldsubstrate surface.

E. Arrays

In some embodiments where the virus recognition moiety is apolynucleotide or polypeptide, a plurality of virus recognition moietiesare arrayed on the substrates using photo activated chemistry,microcontact printing, and ink-jet printing. In particularly preferredembodiments, photolithography is utilized (See e.g., U.S. Pat. Nos.6,045,996; 5,925,525; and 5,858,659; each of which is hereinincorporated by reference). Using a series of photolithographic masks todefine substrate exposure sites, followed by specific chemical synthesissteps, the process constructs high-density arrays of oligonucleotides,with each probe in a predefined position in the array. Multiple probearrays are synthesized simultaneously on, for example, a large glasswafer. The wafers are then diced, and individual probe arrays arepackaged in injection-molded plastic cartridges, which protect them fromthe environment and serve as chambers for hybridization.

In other embodiments, nucleic acid virus recognition moieties areelectronically captured on a suitable substrate (See e.g., U.S. Pat.Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics, thistechnology enables the active movement and concentration of chargedmolecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given target areelectronically placed at, or “addressed” to, specific sites on themicrochip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

In still further embodiments, virus recognition moieties are arrayed ona suitable substrate by utilizing differences in surface tension (Seee.g., U.S. Pat. Nos. 6,001,311; 5,985,551; and 5,474,796; each of whichis herein incorporated by reference). This technology is based on thefact that fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on a X/Y translation stage under aset of four piezoelectric nozzles, one for each of the four standard DNAbases. The translation stage moves along each of the rows of the arrayand the appropriate reagent is delivered to each of the reaction site.For example, the A amidite is delivered only to the sites where amiditeA is to be coupled during that synthesis step and so on. Common reagentsand washes are delivered by flooding the entire surface and thenremoving them by spinning.

In still further embodiments, virus recognition moieties are spottedonto a suitable substrate. Such spotting can be done by hand with acapillary tube or micropipette, or by an automated spotting apparatussuch as those available from Affymetrix and Gilson (See e.g., U.S. Pat.Nos. 5,601,980; 6,242,266; 6,040,193; and 5,700,637; each of which isincorporated herein by reference).

E. Blocking

In some embodiments, following immobilization of the recognition moietyon the surface of the substrate, the remainder of the substrate isblocked to guard against non-specific binding to the substrate surface.Examples of suitable blocking agents, include, but are not limited to,serum albumins, zwitterionic polymers, adsorbed lipid layers, dextranand other sugars, cross-linked lipids, polyethylene oxide,polyoxazolines, hydrogels, and milk. In preferred embodiments, theblocking agent bovine serum albumin, human serum albumin or equine serumalbumin.

IV. Mesogens

Any compound or mixture of compounds which forms a mesogenic layer canbe used in conjunction with the present invention. The mesogens can formthermotropic or lyotropic liquid crystals. Both the thermotropic andlyotropic liquid crystals can exist in a number of forms includingnematic, chiral nematic, smectic, polar smectic, chiral smectic,frustrated phases and discotic phases.

TABLE 1 Molecular structure of mesogens suitable for use in LiquidCrystal Assay Devices Mesogen Structure Anisaldazine

NCB

CBOOA

Comp A

Comp B

DB₇NO₂

DOBAMBC

nOm n = 1, m = 4: MBBA n = 2, m = 4: EBBA

nOBA n = 8: OOBA n = 9: NOBA

nmOBC

nOCB

nOSI

98P

PAA

PYP906

nSm

Presently preferred mesogens are displayed in Table 1. In a particularlypreferred embodiment, the mesogen is a member selected from the groupconsisting of 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butlyaniline and combinations thereof.

The mesogenic layer can be a substantially pure compound, or it cancontain other compounds which enhance or alter characteristics of themesogen. Thus, in one preferred embodiment, the mesogenic layer furthercomprises a second compound, for example and alkane, which expands thetemperature range over which the nematic and isotropic phases exist. Useof devices having mesogenic layers of this composition allows fordetection of the analyte recognition moiety interaction over a greatertemperature range.

In some preferred embodiments, the mesogenic layer further comprises adichroic dye or fluorescent compound. Examples of dichroic dyes andfluorescent compounds useful in the present invention include, but arenot limited to, azobenzene, BTBP, polyazo compounds, anthraquinone,perylene dyes, and the like. In particularly preferred embodiments, adichroic dye of fluorescent compound is selected that complements theorientation dependence of the liquid crystal so that polarized light isnot required to read the assay. In some preferred embodiments, if theabsorbance of the liquid crystal is in the visible range, then changesin orientation can be observed using ambient light without crossedpolars. In other preferred embodiments, the dichroic dye or fluorescentcompound is used in combination with a fluorimeter and the changes influorescence are used to detect changes in orientation of the liquidcrystal.

V. Direct Detection of Entities with Lipid Membranes

The present invention provides methods and devices for the directdetection of entities having a biological membrane, including virusesand bacteria that are pathogens. The systems and devices of the presentinvention can be of any configuration that allows for the contact of amesogenic layer with an organic layer or inorganic layer (e.g., metal,metal salt or metal oxide). The only limitations on size and shape arethose that arise from the situation in which the device is used or thepurpose for which it is intended. The device can be planar ornon-planar. Thus, it is within the scope of the present invention to useany number of polarizers, lenses, filters lights, and the like topractice the present invention.

The systems and devices of the present invention find use in thedetection of variety of viruses and entities having lipid membranes.Examples of such entities having lipid membranes include, but are notlimited to, viruses, bacteria, liposomes, cells, mycoplasmas,protozoans, fungi and the like.

The present invention is not limited to the detection of any particulartype of virus. Indeed, the present invention contemplates the detectionof a variety of viruses, including viruses from the following families:Adenoviridae, Arenaviridae, Astroviridae, Birnaviridae, Bunyaviridae,Caliciviridae, Circoviridae, Coronaviridae, Filoviridae, Flaviviridae,Hepadnaviridae, Herpesviridae, Iridoviridae, Filoviridae,Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae,Picornaviridae, Poxyiridae, Reoviridae, Retroviridae, Rhabdoviridae,Togaviridae, Badnavirus, Bromoviridae, Comoviridae, Geminiviridae,Partitiviridae, Potyviridae, Sequiviridae, and Tombusviridae; thefollowing genera: Mastadenovirus, Aviadenovirus, African swinefever-like viruses, Arenavirus, Arterivirus, Astrovirus, Aquabimavirus,Avibirnavirus, Bunyavirus, Hantavirus, Nairovirus, Phlebovirus,Calicivirus, Circovirus, Coronavirus, Torovirus, Deltavirus, Filovirus,Flavivirus, Japanese Encephalitis Virus group, Pestivirus, HepatitisC—like viruses, Orthohepadnavirus, Avihepadnavirus, Simplexvirus,Varicellovirus, Cytomegalovirus, Muromegalovirus, Roseolovirus,Lymphocryptovirus, Rhadinovirus, Ranavirus, Lymphocystivirus, Goldfishvirus-like viruses, Influenzavirus A, B, Influenzavirus C, Thogoto-Likeviruses, Polyomavirus, Papillomavirus, Paramyxovirus, Morbillivirus,Rubulavirus, Pneumovirus, Parvovirus, Erythrovirus, Dependovirus,Enterovirus, Rhinovirus, Hepatovirus, Cardiovirus, Aphthovirus,Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus,Suipoxvirus, Molluscipoxvirus, Yatapoxvirus, Orthoreovirus, Orbivirus,Rotavirus, Coltivirus, Aquareovirus, mammalian type B retroviruses,mammalian type C retroviruses, avian type C retroviruses, type Dretroviruses, blv-htlv retroviruses, Lentivirus, Spumavirus,Vesiculovirus, Lyssavirus, Ephemerovirus, Alphavirus, Rubivirus,Badnavirus, Alfamovirus, Ilarvirus, Bromovirus, Cucumovirus, Tospovirus,Capillovirus, Carlavirus, Caulimovirus, Closterovirus, Comovirus,Fabavirus, Nepovirus, Dianthovirus, Enamovirus, Furovirus, Subgroup IGeminivirus, Subgroup II Geminivirus, Subgroup III Geminivirus,Hordeivirus, Idaeovirus, Luteovirus, Machlomovirus, Marafivirus,Necrovirus, Partitiviridae, Alphacryptovirus, Betacryptovirus,Potexvirus, Potyvirus, Rymovirus, Bymovirus, Fijivirus, Phytoreovirus,Oryzavirus, Nucleorhabdovirus, Sequivirus, Waikavirus, Sobemovirus,Tenuivirus, Tobamovirus, Tobravirus, Carmovirus, Tombusvirus,Trichovirus, Tymovirus, Umbravirus; and the following species: humanadenovirus 2, fowl adenovirus 1, African swine fever virus, lymphocyticchoriomeningitis virus, equine arteritis virus, human astrovirus 1,infectious pancreatic necrosis virus, infectious bursal disease virus,Bunyamwera virus, Hantaan virus, Nairobi sheep disease virus, sandflyfever Sicilian virus, vesicular exanthema of swine virus, chicken anemiavirus, avian infectious bronchitis virus, Berne virus, hepatitis deltavirus, Marburg virus, yellow fever virus, west Nile virus, bovinediarrhea virus, hepatitis C virus, hepatitis B virus, duck hepatitis Bvirus, human herpesvirus 1, human herpesvirus 3, human herpesvirus 5,human cytomegalovirus, mouse cytomegalovirus 1, human herpesvirus 6,human herpesvirus 4, ateline herpesvirus 2, frog virus 3, floundervirus, goldfish virus 1, influenza A virus, influenza B virus, influenzaC virus, Thogoto virus, murine polyomavirus, cottontail rabbitpapillomavirus (Shope), Paramyxovirus, human parainfluenza virus 1,measles virus, mumps virus, human respiratory syncytial virus, miceminute virus, B19 virus, adeno-associated virus 2, poliovirus 1, humanrhinovirus 1A, porcine rhinovirus, hepatitis A virus,encephalomyocarditis virus, St. Louis encephalomyocarditis virus,foot-and-mouth disease virus O, vaccinia virus, orf virus, fowlpoxvirus, sheeppox virus, monkey pox virus, myxoma virus, swinepox virus,Molluscum contagiosum virus, Yaba monkey tumor virus, reovirus 3,bluetongue virus 1, simian rotavirus SA11, Colorado tick fever virus,golden shiner virus, mouse mammary tumor virus, murine leukemia virus,avian leukosis virus, Mason-Pfizer monkey virus, bovine leukemia virus,human immunodeficiency virus 1, human spumavirus, vesicular stomatitisIndiana virus, rabies virus, bovine ephemeral fever virus, Sindbisvirus, rubella virus, commelina yellow mottle virus, alfalfa mosaicvirus, tobacco streak virus, brome mosaic virus, cucumber mosaic virus,tomato spotted wilt virus, apple stem grooving virus, carnation latentvirus, cauliflower mosaic virus, beet yellows virus, cowpea mosaicvirus, broad bean wilt virus 1, tobacco ringspot virus, carnationringspot virus, pea enation mosaic virus, soil-borne wheat mosaic virus,maize streak virus, beet curly top virus, bean golden mosaic virus,barley stripe mosaic virus, raspberry bushy dwarf virus, barley yellowdwarf virus, maize chlorotic mottle virus, maize rayado fino virus,tobacco necrosis virus, white clover cryptic virus 1, white clovercryptic virus 2, potato virus X, potato virus Y, ryegrass mosaic virus,barley yellow mosaic virus, Fiji disease virus, wound tumor virus, riceragged stunt virus, potato yellow dwarf virus, tobacco necrosissatellite, parsnip yellow fleck virus, rice tungro spherical virus,Southern bean mosaic virus, rice stripe virus, tobacco mosaic virus,tobacco rattle virus, carnation mottle virus, tomato bushy stunt virus,apple chlorotic leaf spot virus, turnip yellow mosaic virus, carrotmottle virus.

The present invention is not limited to the detection of any particulartype of bacteria. Indeed, the detection of variety of bacteria iscontemplated, including, but not limited to Gram-positive cocci such asStaphylococcus aureus, Streptococcus pyogenes (group A), Streptococcusspp. (viridans group), Streptococcus agalactiae (group B), S. bovis,Streptococcus (anaerobic species), Streptococcus pneumoniae, andEnterococcus spp.; Gram-negative cocci such as Neisseria gonorrhoeae,Neisseria meningitidis, and Branhamella catarrhalis; Gram-positivebacilli such as Bacillus anthracis, Bacillus subtilis, Corynebacteriumdiphtheriae and Corynebacterium species which are diptheroids (aerobicand anerobic), Listeria monocytogenes, Clostridium tetani, Clostridiumdifficile, Escherichia coli, Enterobacter species, Proteus mirablis andother spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Campylobacterjejuni, Legionella peomophilia, Mycobacterium tuberculosis, Clostridiumtetani, Hemophilus influenzae, Neisseria gonorrhoeae, Treponemapallidum, Bacillus anthracis, Vibrio cholerae, Borrelia burgdorferi,Cornebacterium diphtheria, Staphylococcus aureus, Bacillus anthracis,and other members of the following genera: Vibrio, Salmonella, Shigella,Pseudomonas, Actinomyces, Aeromonas, Bacillus, Bacteroides, Bordetella,Brucella, Campylobacter, Capnbocylophaga, Clamydia, Clostridium,Corynebacterium, Eikenella, Erysipelothriz, Escherichia, Fusobacterium,Hemophilus, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium,Mycoplasma, Neisseria, Nocardia, Pasteurella, Proteus, Pseudomonas,Rickettsia, Salmonella, Selenomonas, Shigelia, Staphylococcus,Streptococcus, Treponema, Bibro, and Yersinia. Bacterial infectionsresult in diseases such as bacteremia, pneumonia, meningitis,osteomyelitis, endocarditis, sinusitis, arthritis, urinary tractinfections, tetanus, gangrene, colitis, acute gastroenteritis,bronchitis, and a variety of abscesses, nosocomial infections, andopportunistic infections.

The present invention is not limited to the detection of any particularfungi. Examples of fungi include, but are not limited to, dermatophytes(e.g., Microsporum canis and other M. spp.; and Trichophyton spp. suchas T. rubrum, and T. mentagrophytes), yeasts (e.g., Candida albicans, C.Tropicalis, or other Candida species), Saccharomyces cerevisiae,Torulopsis glabrata, Epidermophyton floccosum, Malassezia furfur(Pityropsporon orbiculare, or P. ovale), Cryptococcus neoformans,Aspergillus fumigatus, Aspergillus nidulans, and other Aspergillus spp.,Zygomycetes (e.g., Rhizopus, Mucor), Paracoccidioides brasiliensis,Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis,and Sporothrix schenckii. Fungal infections (mycoses) may be cutaneous,subcutaneous, or systemic. Superficial mycoses include tinea capitis,tinea corporis, tinea pedis, onychomoycosis, perionychomycosis,pityriasis versicolor, oral thrush, and other candidoses such asvaginal, respiratory tract, biliary, eosophageal, and urinary tractcandidoses. Systemic mycoses include systemic and mucocutaneouscandidosis, cryptococcosis, aspergillosis, mucormycosis (phycomycosis),paracoccidioidomycosis, North American blastomycosis, histoplasmosis,coccidioidomycosis, and sporotrichosis. Fungal infections alsocontribute to meningitis and pulmonary or respiratory tract diseases.Opportunistic fungal infections have proliferated, particularly inimmunocompromised patients such as those with AIDS. Preferred organismsinclude Escherichia coli, Streptococcus pneumoniae, Staphylococcusaureus, Saccharomyces cerevisiae, Aspergillus fumigatus, and Aspergillusnidulans. See Goodman and Gilman's Pharmacological Basis ofTherapeutics, (8th ed., 1990) Table 44-1, page 1024-1033, for additionalmicrobial pathogens, diseases, and current therapeutic agents. Theabove-described cells are generally available, for example, from theAmerican Type Culture Collection.

The present invention is not limited to the detection of any particulartypes of cells. Examples of such cells include, but are not limited to,Chinese hamster ovary cells (CHO-K1, ATCC CC1-61); bovine mammaryepithelial cells (ATCC CRL 10274; bovine mammary epithelial cells);monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture; see, e.g., Graham et al., J. Gen Virol., 36:59[1977]); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertolicells (TM4, Mather, Biol. Reprod. 23:243-251 [1980]); monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TR1 cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 [1982]); MRC5 cells; FS4 cells; rat fibroblasts (208F cells); MDBK cells (bovinekidney cells); human hepatoma line (Hep G2), and, for example, thefollowing cancerous cells or cells isolated from the followingcarcinomas: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, Ewing's tumor, lymphangioendotheliosarcoma,synovioma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilns' tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma; leukemias, acute lymphocyticleukemia and acute myelocytic leukemia (myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia); chronic leukemia(chronic myelocytic (granulocytic) leukemia and chronic lymphocyticleukemia); and polycythemia vera, lymphoma (Hodgkin's disease andnon-Hodgkin's disease), multiple myeloma, Waldenstrbm'smacroglobulinemia, and heavy chain disease.

Accordingly, in some embodiments, the present invention providessubstrates comprising at least one detection region comprising arecognition moiety that binds to or otherwise interacts with a virus ora biological entity having a lipid membrane. In preferred embodiments,the detection regions are discreet and created by arraying at least onerecognition moiety on the surface of the substrate. As described above,the inventors have made the surprising discovery that viral particlesbound to a virus recognition moiety on a substrate surface provide forthe homeotropic orientation of mesogens in a liquid crystal independentof the presence of any other homeotropic director (e.g., surfacetopography that causes homeotropic orientation) in the detection region.Also, the inventors have surprisingly found that entities with lipidmembranes (e.g., cells) also provide for the homeotropic orientation ofmesogens independent of the presence of other homeotropic directors.Accordingly, in some preferred embodiments, the detection region doesnot include structures which homeotropically orient mesogens in a liquidcrystal in the absence of virus or entity with a lipid membrane bound toor otherwise interacting with the detection region. In preferredembodiments, the recognition moiety is immobilized on the substrate asdescribed in detail above. In some embodiments, a plurality ofrecognition moieties are arrayed on the surface of the substrate so thatmultiplexed assays for a variety of viruses and/or entities having alipid membrane can be performed simultaneously. In other embodiments,the control regions are included on the substrate that comprise controlspecies immobilized on the surface of the substrate or which provide asite to contact with a control sample containing a known amount of theentity that is being detected.

The present invention is not limited to any particular method ofdetection a change in the orientation of the mesogens in the device.Thus, it is within the scope of the present invention to use lights,microscopes, spectrometry, electrical techniques and the like to aid inthe detection of a change in the mesogenic layer. In those embodimentsutilizing light in the visible region of the spectrum, the light can beused to simply illuminate details of the mesogenic layer to provide forvisual detection. Alternatively, the light can be passed through themesogenic layer and the amount of light transmitted, absorbed orreflected can be measured. The device can utilize a backlighting devicesuch as that described in U.S. Pat. No. 5,739,879. Light in theultraviolet and infrared regions is also of use in the presentinvention. Microscopic techniques can utilize simple light microscopy,confocal microscopy, polarized light microscopy, atomic force microscopy(Hu et al., Langmuir 13:5114-5119 (1997)), scanning tunneling microscopy(Evoy et al., J. Vac. Sci. Technol A 15:1438-1441, Part 2 (1997)), andthe like. Spectroscopic techniques of use in practicing the presentinvention include, for example, infrared spectroscopy (Zhao et al.,Langmuir 13:2359-2362 (1997)), raman spectroscopy (Zhu et al., Chem.Phys. Lett. 265:334-340 (1997)), X-ray photoelectron spectroscopy (Jianget al., Bioelectroch. Bioener. 42:15-23 (1997)) and the like. Visibleand ultraviolet spectroscopies are also of use in the present invention.Other useful techniques include, for example, surface plasmon resonance(Evans et al., J. Phys. Chem. B 101:2143-2148 (1997), ellipsometry(Harke et al., Thin Solid Films 285:412-416 (1996)), electrical methods(such as impedometric methods (Rickert et al., Biosens. Bioelectron.11:757:768 (1996)), and the like.

In some embodiments, the devices of the present invention furthercomprise an electrode or series of electrodes. In some preferredembodiments, at least two electrodes are provided in a plane on one ofthe surfaces of the device substrate. A variety of electrodes may beutilized, including, but not limited to, interdigitated, hyperbolic,triangular and rectangular electrodes. In some particularly preferredembodiments, the device comprises interdigitated electrodes. FIGS. 3 aand 3 b provide a schematic depiction of a device and preferredelectrodes of the present invention. FIG. 3 a depicts liquid crystalmolecules supported on a nanostructured surface coated with arecognition moiety (in this embodiment an antibody). In the absence ofbound analyte, the mesogens assume a planar orientation. Upon binding ofan analyte (in this embodiment, virus particles) on to the surface themolecules align perpendicular to the surface inducing a change in thecapacitance between two electrodes. FIG. 3 b present a schematic ofinterdigitated electrodes. In this embodiment, the size of the arrows ison the order of 500 μm. FIG. 7 presents a schematic depiction of asubstrate configured for dielectrophoresis. The electrode is formed onthe surface of the substrate by methods known in the art (e.g.,photolithography, printing, etc.). The electrode includes a circuit thatinterfaces with power source (e.g., an alternating current source) and aphase inverter. In some embodiments, a mask (e.g., formed from PDMS) isused to contain the sample on the substrate during dielectrophoresis.

In preferred embodiments, the electrodes are utilized to transfer viralor other particles to a surface of the assay device, preferably to asurface comprising recognition moieties. The electrodes are alsoutilized to measure changes in dielectric capacitance of the device(described in more detail below and in the examples).

A challenge that confronts the realization of rapid surface-baseddetection systems is the efficient capture of viruses present in theliquid sample by the assay surface. Most approaches rely on diffusion ofvirus to the surface and on enhancement of this process by eithermechanical approaches to increase fluid motion or by increases intemperature. These are satisfactory approaches when sufficient numbersof virus particles are present in a reasonably large volume of sample,and the contact of sample with the surface occurs over reasonably longperiods of time (hours) such as in virus neutralization, plaquereduction neutralization assays or ELISAs.

In some preferred embodiments, the methods of the present inventionutilize dielectrophoresis (DEP) to capture and concentrate virusparticles from biological samples directly onto functionalized assaysurfaces. DEP is the transport of polarizable particles by a non-uniformtime-dependent electric field. The present invention is not limited toany mechanism of action. Indeed, an understanding of the mechanism ofaction is not necessary to practice the present invention. The DEP forceis generated by the interaction of an induced dipole and a non-uniformfield; the strength and magnitude of that field being related to thedielectric properties of the analyte (e.g., viruses) and the ionicstrength of the medium in which the analyte suspended.

Early work on the effects of DEP on biological particles showed that thedielectrophoretic behavior of two viruses, herpes simplex (HSV) andtobacco mosaic virus (TMV) depended on the frequency of the electricfield and the dielectric properties of the virus. At a frequency of 6MHz in a solution with electrolyte conductivity of 10 mSm⁻¹, TMVexperienced positive DEP (movement to regions of the highest electricfields). In contrast, under the same conditions, HSV experiencednegative DEP (movement to regions of the lowest electric field). In1999, Morgan et al., Separation of submicron bioparticles bydielectrophoresis. Biophysical Journal 77: 516-525 (1999), demonstratedthat dielectrophoresis could be used to separate heterogeneous mixturesof viruses. Using microfabricated polynomial electrodes, theysuccessfully separated a mixture of TMV and HSV. Studies have also shownthat the dielectric properties of a virus are affected by changes in thephysical or biochemical makeup of the virus particle, such as mechanicaldamage to the envelope or enzymatic stripping of surface proteins(Hughes et al., Measuring the dielectric properties of herpes simplexvirus type 1 virions with dielectrophoresis. Biochimica et BiophysicaActa 1571: 1-8 (2002). Using vaccinia virus labeled with lipophiliccarbocyanin dyes and nucleophilic Hoechst dyes, Akin et al. Real-timevirus trapping and fluorescent imaging in microfluidic devices, NanoLetters 4: 257-259 (2003) have demonstrated real-time imaging of thecapture and trapping of virus particles by dielectrophoretic filterswithin a microfluidic biochip. In a step towards the development of arapid diagnostic for food-borne pathogens, Suchiro et al. Selectivedetection of specific bacteria using dielectrophoretic impedancemeasurement method combined with an antigen-antibody reaction, Journalof Electrostatics 58: 229-246 (2003) combined measurement of DEPimpedance with antibody agglutination to detect bacteria in suspension.

Forces arising from DEP can be used to rapidly concentrate, manipulate,and even separate viruses from small sample volumes. The experimentsdescribed above, however, were conducted using model systems of veryhigh concentration, purified virus (up to 10¹² pfu/mL) suspended inmedia of very low ionic strength. For practical application to viraldiagnostics, DEP must be utilized under conditions of physiologicalionic strength (600 mSm⁻¹ or greater) and must effectively.

The methods of the present invention contemplate dielectrophoreticforces on viruses to be of the order of 1 pN. This force, when acting ona virus, generates velocities of ˜100 μms⁻¹. Thus, in preferredembodiments, the time taken for the particle to travel a 100 μm distanceis on the order of 1 s. In contrast, Brownian forces acting on virusparticles give rise of diffusion coefficients of ˜10⁻¹² m²s⁻¹. Thus thetime taken by the virus particle to diffuse through the same distance of100 μm in absence of dielectrophoretic force is 1.4 hrs. It is thuscontemplated that in preferred embodiments, dielectrophoretic forces canaccelerate the transport of viruses to surfaces by 3 orders ofmagnitude.

In further preferred embodiments, the present of analyte in a sample isdetermined by measuring the dielectric capacitance of the device. Thepresent invention is not limited to a particular mechanism of action.Indeed, an understanding of the mechanism of action is not necessary topractice the present invention. Nevertheless, it is contemplated thatliquid crystals have large, anisotropic electrical properties that arereflected in changes in electrical capacitance related to orientationwithin an electrical field. The method of the present invention, basedon dielectric transduction, relies on the principle of change incapacitance between two electrodes when dielectric properties of themedium between them changes. Thus, in some embodiments of the presentinvention, DEP is utilized to force an analyte (e.g., virus) to theanalytic surface. When the analyte binds to the surface, it induces achange in the dielectric property of the medium between the electrodes.When this occurs, it is contemplated that only a very small fraction ofthe electric field distribution between electrodes will be affected andthe change in capacitance between the electrodes will be negligiblysmall. However, in preferred embodiments, where a film of liquid crystalis placed over the bound virus, the orientational transition of theliquid crystals in response to the virus is propagated throughout theentire layer of liquid crystals affecting almost the entire electricfield distribution and the change in capacitance is large and measurablewith commercially available devices. It is contemplated the methods ofthe present invention can be utilized to detect fewer than about 10,000analyte particles (e.g., viruses) in a sample, preferably fewer thanabout 1,000 analyte particles in a sample, more preferably fewer thanabout 100 analyte particles in a sample, and most preferably fewer thanabout 10 analyte particles in a sample.

In still further embodiments, the present invention provides devices fordetecting the change in dielectric capacitance. FIG. 14 provides aschematic depiction of such a device. The detection device preferablycomprises a housing configured to receive an assay device. In preferredembodiments, the housing has an opening therein into which the assaydevice is inserted. In further embodiments, insertion of the assaydevice into the detection device causes the electrodes on the device tocontact an oscillator circuit. In some preferred embodiments, amicroprocessor (such as DS1086 ECONOSCILLATOR, MAXIM Integrated ProductsInc.) based oscillator circuit is utilized to generate an AC voltagewith the desired amplitude and frequency output. In preferredembodiments, the frequency of the applied electric fields ranges frombetween about 100 Hz to about 50 MHz. In further preferred embodiments,in order to maintain a small form factor, the input DC voltage issupplied from a battery source. The oscillator circuits provide variableoutput voltage and frequency by adjusting the parameters, such ascapacitance/resistance of the oscillator circuit. In still furtherembodiments, the devices comprise a liquid crystal display to provide aneasy read-out for the output parameters of the oscillator circuit. Insome embodiments, a battery charger is integrated into the device torecharge the battery. In still other preferred embodiments, thedetection devices comprise a microprocessor that measures thedifferential capacitance between the electrodes. In some preferredembodiments, the microprocessor is a MS3110 chip. This chip yields thedifference between the capacitance in the form of output voltage.

The devices of the present invention can be used to detect the presenceof wide variety of biological entities in a sample, including, but notlimited to those described above. Likewise, the devices of the presentinvention can be used to detect biological entities in a variety ofsamples. In some embodiments, the biological sample is a biologicalfluid, tissue homogenate, feces, vesicular fluid, swab of an orifice ortissue, or media in which virus has been cultured or prepared. In someembodiments, the biological fluid is cerebral-spinal fluid, urine,serum, plasma, nasal secretion, sputum, semen or saliva.

Biological samples may be collected by a variety of techniques. In someembodiments, whole blood is collected by one of many routes (e.g.,venipuncture or fingerstick) into a tube containing an anticoagulantsuch as heparin or sodium citrate. The blood is mixed and then a sampleis removed and placed into contact with a sensing surface. In someembodiments, serum is obtained by permitting blood collected asdescribed to form a clot in the tube. The tube is subjected tocentrifugation or is permitted to sit for one or more hours so that theserum component separates from the cellular component. A sample of theserum is placed in contact with the sensing surface. In someembodiments, tissue homogenates are utilized. Pieces of organs (e.g.,kidney, spleen, heart, brain, liver, lymph nodes) are either minced byscissors or blades or are placed into a container with fluid (PBS, otherbuffers, media, water, etc) and homogenized using a plastic pestle or byinsertion of a mechanical homogenizer into the container until there areno large pieces of tissue visible. The preparation is centrifuged at lowspeed (<20,000 rpm for 5-60 minutes) to remove the particulate materialremaining. The supernatant is placed in contact with the devicesubstrate surface. In some embodiments, spinal fluid is collected fromthe spinal cord by a needle. The fluid is inserted into a sterile tube.A sample of the spinal fluid is placed into contact with the devicesubstrate surface. In some embodiments, a sample of nasal secretions iscollected onto a cotton or synthetic applicator swab and the swab isplaced into a fluid (PBS, water, media, other buffers etc). An aliquotof the sample is placed in contact with the device substrate surface. Insome embodiments, a nasopharyngeal aspirate sample is collected byinsertion of the swab into the nasopharynx. The swab is placed into atube containing fluid (PBS, media, water, buffers) and a sample of thefluid is placed in contact with the device substrate surface. In someembodiments, the biological sample is obtained from an intermediate hostanimal (e.g., a mosquito in the case of West Nile Virus). One or morethan one mosquito is suspended in liquid such as phosphate bufferedsaline or other buffers or media used to grow cells in culture or water.The mosquitoes are homogenized by use of a disposable plastic pestle orby insertion of a mechanical homogenizer into the container. Themosquitoes are homogenizes until no intact insects are visible. Thehomogenate is subjected to a low speed centrifugation (e.g., 2,000 rpmfor 5 minutes) and the supernatant is collected. The supernatant isplaced into contact with a device substrate surface. In any of theforegoing embodiments, the sample may require additional centrifugationif particulate matter is visible.

In some embodiments, a second substrate is provided which is configuredopposite the first substrate so that cell is formed. In someembodiments, the second substrate is also arrayed with recognitionmoieties, while in other embodiments, the second substrate is free ofrecognition moieties. In some embodiments, the second substrate isblocked to prevent non-specific binding or resists non-specific binding.

In some embodiments, samples suspected of containing a virus or entityhaving a lipid membrane are allowed to contact a detection region(s) onthe first substrate. The sample is allowed to contact the substrate fora period of time (e.g., for about 0.5-24 hours, preferably about 2 to 10hours, and most preferably about 1.5 to 5 hours). In some embodiments,the substrate is rocked during the incubation period. In someembodiments, flowing incubation, the substrate is washed with a suitablebuffer (e.g., PBS). The preceding steps can be performed in the presenceor absence of the second substrate. For example, in some embodiments,the sample is applies to the substrate and the incubation and wash stepsare performed without assembling a cell. In other embodiments, the cellis assembled and the incubation and wash steps are performed in thecell.

Following the wash step, the cell is constructed if necessary. In someembodiments, mesogens are then added to the cell so that a liquidcrystal is formed in the cell. The cell is then incubated for a periodof time to allow for a change to occur in the liquid crystal. In someembodiments, the change in the liquid crystal occurs immediately. Thepresent assays operate a variable temperature range. In someembodiments, the incubation is conducted at about 15 to 50 degrees C.,preferably from about 22 to 35 degrees C.

Following incubation with the liquid crystal, the cell is assayed forwhether a change in the liquid crystal has occurred over one or more ofthe detection regions. Although many changes in the mesogenic layer canbe detected by visual observation under ambient light, any means fordetecting the change in the mesogenic layer can be incorporated into, orused in conjunction with, the device. Thus, it is within the scope ofthe present invention to use lights, microscopes, spectrometry,electrical techniques and the like to aid in the detection of a changein the mesogenic layer. In some embodiments, binding of virus to thevirus recognition moiety is detected by a change in the color andtexture of the liquid crystal. The present invention is not limited toany particular mechanism of action. Indeed, an understanding of themechanism of action is not necessary to practice the invention.Nevertheless, it is believed that the change in color and texture is duetilting of the mesogens in the liquid crystal prior to assumption of ahomeotropic orientation.

Accordingly, in those embodiments utilizing light in the visible regionof the spectrum, the light can be used to simply illuminate details ofthe mesogenic layer. Alternatively, the light can be passed through themesogenic layer and the amount of light transmitted, absorbed orreflected can be measured. The device can utilize a backlighting devicesuch as that described in U.S. Pat. No. 5,739,879, incorporated hereinby reference. Light in the ultraviolet and infrared regions is also ofuse in the present invention.

In some embodiments, the cell is placed in between cross polar lensesand light is passed though the lenses and the cell. Areas of homeotropicorientation appear black, while areas of planar orientation appearbright. Thus, the presence of bound virus is indicated by a black fieldwhile areas where no virus is bound are indicated by a bright field.

In some embodiments, the present invention utilizes plate readers todetect changes in the orientation of mesogens upon binding of ananalyte. In particular, the present invention includes methods andprocesses for the quantification of light transmission through films ofliquid crystals based on quantification of transmitted or reflectedlight.

The present invention is not limited to any particular mechanism ofaction. Indeed, an understanding of the mechanism of action is notrequired to practice the present invention. Nevertheless, it iscontemplated that ordered nanostructured substrates impart order to thinfilms of liquid crystal placed onto their surface. These ordered filmsof liquid crystal preserve the plane of polarized light passed throughthem. If the liquid crystal possesses a well-defined distortion—such asa 90 degree twist distortion-then the liquid crystal will change thepolarization of the transmitted light in a well-defined and predictablemanner. It is further contemplated that ordered films (e.g., areas ofhomeotropic orientation) of liquid crystal differentially absorb(relative to randomly ordered films of liquid crystal) specificwavelengths of light.

Accordingly, the present invention contemplates the use of plate readersto detect light transmission through an LC assay device when viewedthrough cross polars, the transmission of light through an LC assaydevice illuminated with a suitable wavelength of light, or reflection oflight (i.e., polarized light or non-polarized light of specificwavelengths) from the surface of an LC assay device. In particularlypreferred embodiments, plate readers are provided that are designed tobe used in conjunction with LC assays. Other embodiments of the presentinvention provide modified commercially available readers such as ELISAreaders and fluorometric readers adapted to read LC assays.

Non-limiting examples of the plate readers useful in conjunction withthe present invention are provided in U.S. patent application Ser. No.10/227,974, incorporated herein by reference. In some embodiments, twopolarizing filters are placed in the optical pathway of the plate readerin a crossed or parallel polar configuration. One filter is placed onthe emission side of the light path prior to passing through the samplewhile a second polarizing filter is placed on the analyzing side of thelight path after light has passed through the sample but before it iscollected by a sensing devise such as a photodiode or a CCD. An orderedliquid crystal in the LC assay device preserves the plane ofpolarization and the amount of light reaching the light gathering andsensing device is markedly attenuated when viewed through cross polarsor markedly accentuated when viewed through parallel polars. Randomorganization of the liquid crystal of the LC assay device does notpreserve the plane of polarization and the amount of light, passingthrough crossed polars, reaching the light collecting and sensing deviceis relatively unaffected. Accordingly, in preferred embodiments, thebinding of target molecules by the recognition moieties in an LC assaydevice introduces disorder into the overlying thin film of LC thatincreases with the amount of bound target molecule. In otherembodiments, specific bandpass filters are placed on the excitation sideof the light path before light encounters the sample as well as on theemission side of the light path (after light has passed through or isreflected by the sample but before reaching the light collecting andsensing device (e.g., photodiode or CCD). This configuration is usefulfor quantifying both reflected and transmitted light

The present invention also provides LC assay devices configured for usein the plate reader. In preferred embodiments, the LC assay device isformatted or arrayed according to the dimensions of standardcommercially available plates (e.g., 24, 96, 384 and 1536 well plates).In some embodiments, the LC assay device comprises a surface (e.g., asubstrate with recognition moieties attached) that is of proper externaldimensions to be accurately fit into a given commercial reader.

It will also be recognized that the present invention provides an assaysystem comprising a plate reading device and an LC assay device, whereinthe plate reading device and LC assay device are configured so thatlight provided from the plate reading device which is passed through orreflected from at least one surface of the LC assay device is detectedby a detection unit of the plate reading device. Suitable detectingunits include CCDs and photomultiplier tubes.

Commercially available plate readers that may be modified according tothe present invention include, but are not limited, to those availablefrom Nalge Nunc International Corporation (Rochester, N.Y.), GreinerAmerica, Inc. (Lake Mary, Fla.), Akers Laboratories Inc., (Thorofare,N.J.), Alpha Diagnostic International, Inc. (San Antonio, Tex.), andQiagen Inc. (Valencia, Calif.).

VI. Non-Specific Detection Following Specific Capture

In some embodiments, the assays of the present find use for thenon-specific detection of an analyte following specific capture. Inthese embodiments, the analyte is captured by a capture substrate (e.g.,a PDMS stamp or bead) displaying a recognition moiety that interactswith the analyte. The analyte is then transferred to a detectionsubstrate to which the analyte non-specifically binds. The presence ofthe analyte on the second substrate is detected by contacting the secondsubstrate with a liquid crystal. Areas of disorder or order within theliquid crystal are indicative of the presence of analyte. As above, avariety of methods are useful for determining whether there is a changesin the orientation of the mesogens of the device. In some embodiments,the assay devices are configured with electrodes as described above sothat the analyte can be transferred to a surface of the assay device byuse of an electric current (e.g., by dielectrophoresis). The electrodesare also used to measured changes in electrical properties of the device(e.g., dielectric capacitance) as a result of changes in liquid crystalorientation.

In some preferred embodiments, the assays of the present invention areused for the detection of multiple species or genera of animals to apathogenic organism. As a non-limiting example, antibodies specific WestNile Virus have been detected in samples collected from horses, mallardducks, pigeons, rabbits, and mice. It will be recognized that theseassays find use for testing samples from avian species such as crow,blue jay, eagles, sparrows and the more than 150 species of birdspresent in the US that are known to be infected with West Nile Viral,horses, humans, small mammals such as dogs and cats and other companionanimals, rodents such as mice and rats, etc., and other wildlife such asraccoons, skunks, felines, canids, etc.

In some embodiments, surfaces of the detection substrate as describedabove are functionalized for protein binding using the chemistriesdescribed above. In some preferred embodiments, the detection substratesare substrates onto which a metal (e.g., gold) has been obliquelydeposited and functionalized with 4-Aminothiophenol (ATP). In preferredembodiments, it is preferred that the compound used to functionalize thesurface of the detection substrate displays a stronger affinity for theligand (e.g., an antibody) than the ligand displays for its bindingpartner (e.g., the envelope protein E of West Nile Virus).

In some embodiments, a stamp substrate surface is prepared that displaysat least one recognition moiety. A stamp substrate is any substrate thatcan be used to transfer an entity that is covalently or non-covalentlybound to the surface of the stamp substrate to another surface. Examplesof suitable stamp substrates include, but are not limited to, PDMS andother elastomeric materials. In some embodiments, differentconcentrations of the same recognition moiety are arrayed in differentareas of the stamp substrate. In other embodiments, a variety ofdifferent recognition moieties (e.g., envelope proteins from differentenveloped viruses) are arrayed on the stamp substrate surface. In someembodiments, multiple recognition moieties in multiple concentrationsare arrayed on the stamp substrate surface. In other embodiments, acontrol area is included on the stamp substrate surface. The recognitionmoiety (or recognition moieties) is then introduced to the stampsubstrate surface, preferably in an array.

In some embodiments, the stamp substrate surface is a functionalizedsurface so that a covalent chemical bond is formed with the recognitionmoiety. In some preferred embodiments, a PDMS substrate isfunctionalized with disuccinimidyl suberate (DSS). In general, it ispreferred that the compound used to functionalize the surface of thestamp substrate displays a stronger affinity for the recognition moiety(e.g., an envelope protein from a virus) than the recognition moietydisplays for its associated analyte (e.g., an antibody to therecognition moiety, in this case an antigen, present in serum). Examplesof other suitable functionalizing agents includes those that are listedabove.

In some embodiments, the stamp substrate is then exposed to a testsample under conditions such that an analyte (e.g., binding partner ofthe recognition moiety) suspected of being contained in the test sampleis captured by the recognition moiety on the stamp substrate. Inpreferred embodiments, the test sample comprises a body fluid from atest subject. After a period incubation (e.g., 10 minutes to about 10hours), the stamp substrate is washed. In some embodiments, thedetection substrate is then contacted with the stamp substrate underconditions such that the analyte (e.g., an antibody) is transferred fromthe stamp substrate surface to the detection substrate. In preferredembodiments, the compound used to functionalize the surface of thedetection substrate displays a stronger affinity for the analyte thandoes the recognition moiety so that the analyte is detached from therecognition moiety and transferred to the detection substrate.

In still further embodiments, the analyte is captured on a bead thatdisplays a recognition moiety. As described above, the beads may beformed from latex, polymers, agarose, or other materials and in somepreferred embodiments are magnetic. In some embodiments, the analyte isthen transferred to the detection substrate. The transfer may beaccomplished in a variety of ways. In some embodiments, the analyte iseluted from the beads either directly onto the detection substrate oreluted and the transferred to the detection substrate by a method suchas spotting. In other embodiments, the beads exposed to analyte arecontacted with the detection substrate so that the analyte istransferred to the detection substrate. As described above, in someembodiments, the detection substrate surface is functionalized with amoiety with a stronger affinity for the analyte than the recognitionmoiety on the bead so that the analyte is transferred to the detectionsubstrate. In some embodiments, the signal from the analyte is amplifiedby binding one or more additional molecules to the analyte prior toelution. For example, if the analyte used is an antibody, a secondaryanti-species antibody (e.g., and anti-Fc antibody for a particularspecies or rabbit-anti-human antibody, mouse-anti-human antibody,mouse-anti-rabbit antibody, etc.). Enzyme-antibody conjugates, analytespecific second antibodies, gold sol particles and other molecules andmolecule systems may also be utilized. Where nucleic acids are beingdetected, the analyte detection assays outlined herein may follow anamplification method such as PCR.

A variety of detection substrates find use in the assays of the presentinvention, including the functionalized substrates described in detailabove. In some preferred embodiments, the detection substrate comprisesa rubbed polyimide or a polyimide that homeotropically orients a liquidcrystal.

In some embodiments, after transfer of the analyte to the detectionsubstrate, a liquid crystal is applied to the detection substrate sothat the presence of the binding partner on the detection substrate canbe detected. A variety of liquid crystal-forming substances can be used,including those listed above. In some preferred embodiments, 5CB isused. In some embodiments, the detection substrate is used to form anoptical cell with another substrate and the liquid crystal is applied toa chamber formed by the two substrates.

As can be seen, the foregoing methods can be adapted to detect ofvariety of analyte-recognition moiety combinations, includingprotein-protein, protein-nucleic acid, nucleic acid-nucleic-acid, andother molecular interactions. The detection is label free. Thus, it iscontemplated that this system is especially useful for multiplexedassays. As will be appreciated, the capture substrate can befunctionalized with a variety of recognition moieties in an array thatcorresponds to a series of discreet detection regions on the detectionsubstrate. Positive signals on the detection substrate can thus becorrelated with the particular ligand on the stamp substrate. Thus, afirst detection area on the detection substrate can be specific for afirst analyte (e.g., an antibody specific for a particular pathogen), asecond detection area on the detection substrate can be specific for asecond analyte (e.g., an antibody specific for a second pathogen or adifferent antibody specific for the first pathogen to provideconfirmatory results), and so on.

The label free detection possible with the present system providesadvantages over currently used processes such as ELISA. The presentsystem does not require a secondary antibody to detect ligand or antigenspecific antibodies from a test subject. This is important because thepresent system can be utilized to detect antigen/ligand specificantibodies from different species in a single assay because separatesecondary antibodies specific for each species are not required. Thisaspect greatly increases the flexibility of the assays and time neededto respond outbreaks of a disease in a wide or previously unstudiedpopulation of subjects. Furthermore, the present system does not requirea labeling systems such as radioactive, fluorescent, or enzymaticsystem. These systems are often relatively unstable or have short shelflives and require specialized equipment (scintillation counters, film)that is not readily adaptable to field use.

VII. Detection with Lipid Tags

In some embodiments, the ability of lipids such as liposomes to orientliquid crystals is utilized to detect an analyte. As described above,the present invention comtemplates the use of recognition moieties orligands that are complexed with lipids. In some embodiments, these lipidcomplexes (e.g., liposomes) are utilized to detect the presence of ananalyte in a sample or on substrate. For example, as described above,lipids and lipid containing entities such liposomes can be derivatizedto display a recognition moiety such as a protein or nucleic acid. Asample or substrate onto which a sample has been applied can then becontacted with the lipid-recognition moiety complex so that therecognition moiety binds to otherwise becomes associated with theanalyte. The resulting analyte-recognition moiety-lipid complex can thenbe detected by transferring the complex to a substrate if necessary andthen contacting the substrate with a liquid crystal. The presentinvention is not limited to any particular mechanism of action. Indeed,an understanding of the mechanism of action is not necessary to practicethe present invention. Nevertheless, it is contemplated that the lipidportion of the complex provides homeotropic orientation to the portionof the liquid crystal in contact with the lipid. The homeotropicorientation can be detected by the methods described above. It will berecognized that it is not necessary that the substrate itself orient theliquid crystal. Thus, these assays can utilize low-cost simplesubstrates that do not provide an anisotropic surface or surface that isotherwise derivatized with an organic layer. Of course, the substrateshave anisotropic surfaces or derivatized surfaces as described above ifdesired.

In other embodiments, the lipid-recognition moiety complexes are used assecondary binding agents to detect an analyte-recognition moietycomplex. For example, an analyte may first be contacted with a firstrecognition moiety. In some embodiments, the first recognition moiety isa ligand for a second recognition moiety complexed with a lipid. Theanalyte-first recognition moiety complex is then contacted with thesecond recognition moiety-lipid complex so that the second recognitionmoiety binds to the first recognition moiety, thus labeling theanalyte-first recognition moiety complex with the lipid. The presence ofthe lipid can then be detected as described above. In some preferredembodiments, the first recognition moiety is fused to either avidin orbiotin so that a lipid complex comprising either avidin or biotin can beused as the secondary binding agent. In other embodiments, if the firstrecognition moiety is an antibody, the second recognition moiety can beprotein A or an antibody that binds to the first antibody, for example,to the Fc region.

VIII. Kits

In some embodiments, the present invention provides kits for thedetection of analytes. In preferred embodiments, the kits comprise oneor more substrates as described in detail above. In some embodiments,the kits comprise capture and detection substrates. In some preferredembodiments, the capture substrates are beads or stamps. In furtherembodiments, the kits comprise a substrate that can be used inconjunction with the detection substrate to assemble a liquid crystalcell. In some embodiments, the kits comprise a vial containing mesogens.In still other embodiments, the kits comprise at least one vialcontaining a control analyte or analytes. In still other embodiments,the kit comprises instructions for using the reagents contained in thekit for the detection of at least one type of analyte. In someembodiments, the instructions further comprise the statement of intendeduse required by the U.S. Food and Drug Administration (FDA) in labelingin vitro diagnostic products. The FDA classifies in vitro diagnostics asmedical devices and requires that they be approved through the 510(k)procedure. Information required in an application under 510(k)includes: 1) The in vitro diagnostic product name, including the tradeor proprietary name, the common or usual name, and the classificationname of the device; 2) The intended use of the product; 3) Theestablishment registration number, if applicable, of the owner oroperator submitting the 510(k) submission; the class in which the invitro diagnostic product was placed under section 513 of the FD&C Act,if known, its appropriate panel, or, if the owner or operator determinesthat the device has not been classified under such section, a statementof that determination and the basis for the determination that the invitro diagnostic product is not so classified; 4) Proposed labels,labeling and advertisements sufficient to describe the in vitrodiagnostic product, its intended use, and directions for use. Whereapplicable, photographs or engineering drawings should be supplied; 5) Astatement indicating that the device is similar to and/or different fromother in vitro diagnostic products of comparable type in commercialdistribution in the U.S., accompanied by data to support the statement;6) A 510(k) summary of the safety and effectiveness data upon which thesubstantial equivalence determination is based; or a statement that the510(k) safety and effectiveness information supporting the FDA findingof substantial equivalence will be made available to any person within30 days of a written request; 7) A statement that the submitterbelieves, to the best of their knowledge, that all data and informationsubmitted in the premarket notification are truthful and accurate andthat no material fact has been omitted; 8) Any additional informationregarding the in vitro diagnostic product requested that is necessaryfor the FDA to make a substantial equivalency determination. Additionalinformation is available at the Internet web page of the U.S. FDA.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); gmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); 1 or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); C(degrees Centigrade); U (units), mU (milliunits); min. (minutes); sec.(seconds); % (percent); kb (kilobase); bp (base pair); PCR (polymerasechain reaction); BSA (bovine serum albumin).

Example 1 Immobilization of Antibodies on Substrates

This example describes different methods for immobilizing antibodies onsubstrate. Five different immobilization strategies were evaluated:

1) HEXA: adsorption of Protein A, then the West Nile Virus monoclonalantibodies (WNV Mabs) onto a hydrophobic monolayer formed fromCH₃(CH₂)₁₅SH (HEXA) on the surface of a gold film. The surface wasblocked with BSA after immobilization of the antibody.

2) SPDP: covalent attachment of WNV Mabs to a monolayer formed from2-mercaptoethylamine (2-MEA) on a gold film by using thesulfhydryl-reactive (protein) and amine-reactive (monolayer)heterobifunctional cross-linker N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). The surface was blocked with BSAafter immobilization of the antibody.

3) PMPI: covalent attachment of Ras polyclonal antibodies (Ras Pabs) toa monolayer formed from 11-mercaptoundecanol (11-MU) on a gold film byusing a sulfhydryl-reactive (protein) and hydroxyl-reactive (monolayer)heterobifunctional cross-linker N-(p-maleimidophenyl)isocyanate (PMPI).The surface was blocked with BSA after immobilization of the antibody.

4) DSS: covalent attachment of Ras Pabs to a monolayer formed from 2-MEAon a gold film by using an amino-reactive homobifunctional cross-linkerdisuccinimidyl suberate (DSS). The surface was blocked with BSA afterimmobilization of the antibody.

5) Adsp.: direct adsorption of WNV Mabs onto a thin film of polyurethanespin coated on the surface of gold. The surface was blocked with BSAafter immobilization of the antibody.

The ellipsometric thicknesses of antibodies immobilized by these fivemethods was analyzed. All five methods resulted in immobilization ofantibodies on surfaces. The three strategies for the covalentimmobilization of antibodies to the surfaces (SPDP, PMPI and DSS) led toapproximately the same level of immobilization of antibodies (anellipsometric thickness of 3 nm). Passive adsorption of antibodies(Adsp) on polyurethane surfaces led to capture of an ellipsometricthickness of 8 nm of antibody, and the Protein A mediated attachment ofantibodies (HEXA) lead to the immobilization of an ellipsometricthickness of ˜4 nm of IgG. The direct adsorption approach (Adsp) andprotein A-mediated adsorption lead to the highest levels of antibodyimmobilization.

Example 2 Detection of West Nile Virus

The detection of viruses with liquid crystal assays in which thedetection region comprises topographical features has been described inWO 01/61357. These types of surfaces in combination with liquid crystalswere successfully used to report the presence of West Nile Virus (WNV)captured on the surface of such substrates. Surprisingly, however, ithas now been found that the reporting mechanism does not require thetopography on the surface. This unexpected outcome substantiallysimplifies the fabrication of substrates for detection of viruses usingliquid crystals. As described below, it has been demonstrated that thisreporting mechanism can be applied to different viruses.

Antibodies to WNV were deposited onto the surface of molded polyurethanereplicas. The micromolded replicas had a pitch of 400 nm and a depth of54 nm. A drop of aqueous solution containing WNV was deposited onto thesurface of the polymeric replica. The solutions contained 10^(8,4)pfu/0.2 ml of WNV and the incubation was performed for 4.5 hours.Following incubation of the droplet containing WNV on the surface, thesurface was rinsed with PBS and then imaged by using liquid crystal.Surprisingly, regions of the surface that were contacted with thesolution containing WNV caused homeotropic anchoring of the liquidcrystal. In particular, the homeotropic region was seen both on theareas of the replica that possess topography as well as the nominallysmooth areas. Because the fabrication of substrates that do not possessa controlled topography is simple and straightforward to perform, thepossibility of using surfaces without topography as the basis of assaysfor viruses using liquid crystals was investigated. As described below,the homeotropic response to WNV is unique to the presence ofspecifically captured virus on the surface.

Next, it was necessary to confirm that the observed response in thetopography-free region was a response to specifically captured WNV onthe surface. Antibodies to SLE, Dengue, LACV and WNV were deposited onthe same planar substrate. WNV stock was rocked across the four regionsfor 17 hours at 35° C. The cell was then observed through crossed polarlenses. The Dengue and LaCV antibody regions displayed brightly coloredand disordered LC, indicating no binding of the WNV to non-specificantibodies. The WNV antibody region displayed a complete homeotropiccircle, indicating bound virus. In the SLE antibody region, there was asmall area of homeotropic alignment, signifying a slight cross-reactionof the WNV to the SLE antibodies.

Several observations were made. First, the homeotropic response of theliquid crystal to WNV is striking and unambiguous. The entire region ofthe surface that was exposed to the droplet containing WNV assumed ahomeotropic orientation. Second, in contrast to the region of thesurface presenting antibodies to WNV, the regions presenting antibodiesto SLE, Dengue and LACV did not cause a homeotropic orientation of theliquid crystal. It is noted that a small area of the surface presentingthe antibodies to SLE did cause homeotropic alignment, however, it isvery small as compared to the area presenting antibodies to WNV, and itwas clearly distinguishable from the area presenting antibodies to WNV.In summary, these results clearly demonstrate that the homeotropicresponse of the liquid crystal to WNV is a response to virus that isspecifically captured on the regions of the surface presentingantibodies to WNV.

These results were obtained using an optical cell that comprised asecond surface of glass treated with OTS. Because the liquid crystalorients homeotropically on the OTS-treated glass surface, thepossibility that the homeotropic response of the liquid crystal to boundvirus was caused by the OTS-treated glass slide in combination with alack of orienting influence of a virus-decorated surface was considered.To address this possibility, an optical cell was prepared from twopolyurethane surfaces decorated with virus. Homeotropic anchoring of theliquid crystal was observed when virus was captured by the antibodies onthe polyurethane surface. From this result it can be concluded that thehomeotropic orientation of the liquid crystal on the virus-decoratedsurfaces is a response to WNV and not caused by the OTS-coated glassslide. Additionally, a planar gold substrate was functionalized withC₁₆SH, then WNV monoclonal antibodies and then treated with WNV. Theplanar gold substrate was then paired with an OTS slide to create anoptical cell. It was observed that homeotropic orientation of the liquidcrystal can be obtained when the antibodies to the WNV are immobilizedon gold films made hydrophobic with hexadecanethiol.

Example 3 Optimization of Antibody Immobilization

Different methods of immobilizing the antibody were investigated todetermine the procedure which would give the best results. Briefly,polyurethane substrates were functionalized with (a) 1 uM WNV monoclonalantibodies, (b) 5 uM WNV monoclonal antibodies, and (c) 1 mg/ml ProteinA first, then 1 uM WNV monoclonal antibodies. All functionalizedsubstrates were then incubated with the WNV stock. The results showedthat substantially the same homeotropic response is observed when thepolyurethane is functionalized with 1 uM or 5 uM antibody, and also whenthe substrate is first incubated with Protein A (molecule whichcorrectly orients the antibody), and then functionalized with 1 uMantibody. These results indicate that a strong homeotropic response canbe obtained with a lower concentration of antibody, with or withoutProtein A. The current method involves coating the entire substrate with1 mg/ml Protein A, and subsequently immobilizing antibodies in specificdetection regions on the substrate.

Example 4 Optimization of Delivery of Virus

The results described above were obtained by incubation of the surfaceswith the virus solution for up to 20 hrs. Next, ways of optimize virusdelivery to the surface-immobilized WNV monoclonal antibodies and thusminimize the binding times were investigated. The following threeparameters were investigated: a) the temperature of the incubation; b)rocking of the sample back-and-forth to generate convection; and (c) theinfluence of the antibody spot size.

First, increasing the incubation temperature to 35 degrees from roomtemperature (approximately 22 degrees C.) decreased the required bindingtime. Next, by delivering an approximately 20 μl drop to the antibodyfunctionalized surface, it was possible to shorten the time needed for a100% homeotropic alignment response from overnight (16-20 hrs) to 3 hrs.To reduce the binding time even further, a ‘rocking’ method wasintroduced into the procedure. Instead of incubating the WNV as astationary 20 μl drop on the substrate, a PDMS “pool” was used tocontain a larger volume (300 μl) of WNV on top of the substrate. The WNVwas then rocked continuously using a bi-directional rotator. This changein procedure led to a reduction of binding time from 3 hours to 2 hours.To determine the effect of reducing the spot size of the antibody, theantibody volume (to be immobilized) was decreased from 20 μl to 5 μl. Bycombining these three methods, increasing temperature, rocking, andreducing the surface area of immobilized antibody, the binding time wasdecreased from 2 hours to 1.5 hours. It should be emphasized that thisis the only incubation time required for the assay. Once the virus andantibody have interacted, the liquid crystals are added and they assumetheir preferred orientation within seconds. Read out is immediate. Theorientation is stable and the assay need not be read immediately. Thisdoes not represent a fixed time requirement. These end points were setas 100% homeotropic alignment. Partial alignment has been noted beforethe 1.5 hr point. These results provide unambiguous evidence that liquidcrystals can be used to detect WNV bound to a substrate coated withantibodies to WNV via the homeotropic response of the liquid crystal.

Example 5 Demonstration of Detection of St. Louis Encephalitis Virus(SLE)

To assess the generality of the method of reporting viruses other thanWNV via the homeotropic response of the liquid crystal, an experimentwas performed to determine if SLE could be detected via the response ofliquid crystal to SLE captured on a surface presenting antibodies toSLE. Assays for SLE were performed essentially as described above forWNV. In each case when SLE virus was tested in the assay, areas ofhomeotropic alignment were observed on the edge of the slide, outside ofthe diagnostic zone but in the direction of the PBS wash. It appears asthough the antigen-antibody complexes that formed on the diagnosticsurface were washed away during the PBS rinse and only some residualremained near the edge of the slide to be visualized by the homeotropicalignment of the liquid crystals. This did not happen with the WNVassay. These areas indicate that SLE can be detected via the homeotropicresponse of the liquid crystal. This result is important, because itsuggests that the homeotropic response of liquid crystal is notrestricted to WNV but can be exploited for detection of viruses otherthan WNV. It would be valuable in times such as this when outbreaks ofWNV, SLE and EEE are occurring simultaneously to have a rapidmulti-plexed assay available.

Example 6 Assays With Multiple Detection Regions

A useful format for assays for viruses is a multi-array with antibodiesto several viruses patterned spatially on the same surface. Todemonstrate the feasibility of patterning antibodies on surfaces, anddetecting the binding of virus to them, an experiment was performed inwhich antibodies to WNV were patterned on three regions of a surface.Briefly, a polyurethane substrate was functionalized with WNV monoclonalantibodies in a lane format. WNV was rocked over the substrate for 2hours at 35° C. Three homeotropic lanes were observed, indicating thelocation of the bound virus.

Example 7 Detection of WNV from Tissue of Birds

This example describes the detection of WNV from the tissue of birds.Polyurethane substrates were prepared by incubation of the surface with1 mg/ml Protein A. Next, 4 droplets containing 1 uM WNV monoclonalantibodies were immobilized within each of several sample areas. Crowsamples comprising kidney/spleen tissue suspensions were obtained fromNWHC's Diagnostic Virology lab. These samples were determined to bepositive or negative by real time RT-PCR, or by tissue cultureisolation, respectively. Tissue suspensions were centrifuged for 10 minat 2,000 rpm, and the supernatant was applied directly to the sampleareas. The WNV stock used in these experiments 10^(7,7) pfu/0.2 ml.Incubation was overnight at 35° C. The polyurethane substrate which wasfunctionalized with WNV monoclonal antibodies was treated with positiveor negative American crow tissue suspensions, culture media, and WNVstock as a positive control. The positive crow and the WNV sampleregions displayed homeotropic alignment, indicating the presence ofbound West Nile virus. The negative crow and culture media sampleregions displayed disordered and brightly colored LC, indicating thelack of bound virus. We note that the positive control and negativecontrols yielded the correct response of the liquid crystal.

Additional control experiments were performed using whole blood frombirds and horses. Chicken blood (with either heparin or citrate as ananti-coagulant) and horse blood was applied directly to the substratewhich was functionalized with WNV antibodies. Incubation was overnightat 35° C. The results clearly demonstrated that there is nocross-reaction or non-specific binding with normal avian or equine wholeblood samples. Additional results were obtained with negative sera fromchicken/horse/mallard/or with culture media. Additionally, six mosquitohomogenates from the NY State Dept of Health were that have previouslyshown to be negative for WNV by Taqman PCR were obtained and tested.These homogenates also tested negative with the LC assay and did notshow any evidence of non-specific binding to the surface. Thisexperiment demonstrates the ability to use concentrated biologicalsamples with little processing in the assays of the present invention.

Example 8 Homeotropic Orientation by Cells

This example demonstrates homeotropic orientation by tissue culturecells. Tissue culture cells are allowed to attach to the surface of aglass slide. The surface is washed and mesogens are placed on thesurface. Areas of the surface occupied by cells appear uniformly dark.Areas not occupied by cells regions display disordered and brightlycolored LC.

Example 9 Detection by Stamp Transfer

This example describes the detection of antibodies transferred from astamp substrate comprising a ligand to a detection substrate. To formthe stamp substrate, 10 parts elastomer to 1 part curing agent from theSylgard 184 Elastomer Kit (Dow Corning) is mixed together, degassed in avacuum desiccator, and cured into PDMS at ˜65° C. for 1 hour. The PDMSstamps are cut out from the PDMS cured to a Fisher's Finest GlassMicroscope Slide. The PDMS stamps are then rinsed with ethanol and driedwith nitrogen to clean them. The PDMS stamps are then plasma ashed in anoxygen plasma (200 mTorr O₂ backfill pressure) at 275 Watts for 4minutes to oxidize the surface of the PDMS producing a surface similarto glass. The PDMS stamps are then submerged in a 2% APES/98% dryacetone solution for 2 minutes with constant stirring. PDMS stamps arethen transferred into acetone for 5 minutes with constant stirring. ThePDMS stamps are then removed from the acetone, rinsed with acetone, anddried with nitrogen. The PMDS stamps are then placed in an ovenuncovered for 30 minutes at 100° C. The PDMS stamps are removed from theoven and cooled to room temperature before placing them into a 1 mM DSS(Pierce) solution made from dissolving 74 mg of DSS in 2 ml of DMSOwhich is mixed with 198 ml of dry methanol. Stamps are kept in 20 ml ofthe DSS chemistry for 1 hour with constant stirring. The PDMS stamps arethen removed from the DSS and rinsed with methanol followed by anitrogen stream to dry them. Twenty μL drops of 0.25 μM protein E (L²Diagnostics, LLC) are then incubated on the individual stamps overnightat 4° C. and for 1.5 hours at room temperature.

An obliquely (350) deposited 30 Å Ti/300 Å Au Corning 1737 microscopeslide is placed in 11 mM ATP (4-aminothiophebol, Aldrich) overnight. Thedroplets of protein E are then rinsed off of the stamps with milli-Qwater from a squirt bottle for approximately 15 seconds followed by anitrogen stream to dry the stamps. Twenty μL drops of 20 mg/ml (totalprotein concentration) anti-E rabbit polyclonals (L² Diagnostics, LLC),20 mg/ml negative rabbit serum (Pierce), 1:2 dilution positive horseserum (Cornell university), and 1:2 dilution negative horse serum(Cornell University) are placed on their individual stamps where theprotein E was incubated (dilutions done in PBS). The sera are incubatedat room temperature for 6 hours. The sera are rinsed off the PDMS stampswith 0.5 ml. of 0.01% Triton in PBS followed by a milli-Q water rinsefor 15 seconds from a squirt bottle. The stamps are then dried withnitrogen. The oblique gold-coated slide is removed from the ATP, rinsedwith ethanol, dried with nitrogen, dipped into the 0.1N HCl, dried withnitrogen, dipped again into the 0.1N HCl, and dried with nitrogen.

The PDMS stamps are then gently placed into contact with the ATP treatedoblique gold slide for 1 minute with gentle pressure for 5 seconds atthe beginning and end of contact. The stamps are removed from thesurface. Optical cells are constructed by separating the stamped obliquegold/ATP surface from a Fisher slide that had been vacuum deposited withOTS ((Tridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane, Gelest),with 25 μm Mylar. The optical cells are held together with binder clips.5CB liquid crystal (4-cyano-4′pentyl-1-1′-biphenyl, EM Science) is thenintroduced into the optical cells in its isotropic phase at ˜40° C.Optical cells are then heated at 37° C. until (usually overnight) theliquid crystal is aligned homeotropically everywhere, unless there isdisruption in a circular form from transferred biological agents.

The results are presented in FIG. 2. In this procedure, protein E iscovalently bound to the DSS chemistry on the PDMS stamp. The protein Ein turn captures WNV antibodies if present in the serum incubationdroplet. Even if there are no WNV antibodies in the serum (in this casethe negative serums or controls on the right of FIG. 2) there is assumedto be some non-specific absorption to the PDMS stamp surface, thus theneed for using the Triton rinse to remove non-specific absorbedentities. When the PDMS stamp is brought into contact with the ATPtreated gold surface the protein E stays covalently bound to the DSSchemistry on the stamp and the captured WNV antibodies, if present,transfer to the ATP chemistry due to the physics of a stronger bond.Referring to the pictures in FIG. 2, the two optical cells on the left,as viewed through crossed-polarizers, were stamped with PDMS that hadsera positive for the WNV antibodies incubated on them. The two pictureson the right of FIG. 2 show the control optical cells, as viewed throughcrossed-polarizers, that were stamped with PDMS that had sera negativefor WNV antibodies incubated on them. When WNV antibodies are present,the stamps that captured WNV antibodies on them transfer to the ATPtreated gold causing a circular (from the shape of the incubationdroplet) disruption pattern in the liquid crystal optical cell, seepictures on the left. The control stamps that have not captured antibodydemonstrate homeotropically aligned liquid crystal in the optical cell(see pictures on the right of FIG. 2).

Example 10 Affinity Contact Printing (αCP) to Report a Specific Antibodyfrom a Population

This experiment demonstrates the capture of a specific antibody from amixture of two antibodies in solution. A six button PDMS stamp, see FIG.4, was plasma ashed in an O₂ cloud for 8 minutes to oxidize the surfaceof the PDMS. The stamp was then placed in a 2% APES in dry acetonesolution for 2 minutes while stirring. The stamp was then placed inacetone for 5 minutes while stirring. The stamp was then rinsed withacetone, dried with nitrogen, and placed in a 100° C. oven for 30minutes. The stamp was then placed in a 1 mM DSS solution for 1 hourwhile stirring. The stamp was removed from the DSS, rinsed withmethanol, and dried with nitrogen. On three of the buttons on the sixbutton PDMS α-stamp, 20-μl drops of 0.25 μM protein E were incubatedovernight at 4° C. The remaining three buttons had 20 μl drops of 0.25μM biotinalated BSA incubated on them overnight at 4° C. The proteinswere rinsed off with water and the α-stamp was dried with nitrogen. Twoof the three protein E functionalized buttons and two of the threebiotinalated BSA functionalized buttons had 20 μl drops of a 0.25 μMmixture of anti-biotin and anti-E incubated on them for 6 hours at roomtemperature. The third protein E functionalized button had a 20 μldroplet of 0.25 μM anti-biotin incubated on it for six hours at roomtemperature as a negative control. The third biotinalated BSAfunctionalized button had a 20 μl droplet of 0.25 μM anti-E incubated onit for six hours at room temperature as a negative control. Each of thesix individual buttons were first rinsed with ˜5 drops of 0.01% Tritonin PBS followed by a 15 second rinse with milli-Q water and then driedwith nitrogen. The six button α-stamp was then put into contact with a 1mM ATP treated 350 obliquely deposited Au slide for 1 minute with a fewseconds of gentle pressure at the beginning to insure contact. Theα-stamp was then peeled off and the optical cell was put together withan OTS slide and 5CB liquid crystal. The optical cell was then incubatedat 40° C. for two days to turn the background LC homeotropic. Theresults are provided in the image in FIG. 5.

Analysis of this data reveals two strong positives from the biotinalatedBSA pulling out its specific antibody, anti-biotin, from a mixture andreporting it in the LC optical cell seen via αCP. Both of the protein Ebuttons that had the antibody mixture incubated on them show a positiveresult around the rim of the printing area from the anti-E transferring,but not as strong of a signal as the anti-biotin readouts. Both negativecontrols were blank, which would imply that the four positive controlspulled out their specific antibodies from the antibody mixture andreported them via αCP. The anti-E signals could not be as strong as theanti-biotin signals for several reasons: too long of an incubation whilewaiting for the optical cell background to turn homeotropic causing someof the disruption to wash away, and/or uneven pressure of the α-stampduring printing.

Example 11 Polyimide Surfaces can Host Molecular Interactions

A silicon wafer was scrubbed with 1-Methyl-2-Pyrrolidinone (NMP). Thescrubbed wafer was spin cleaned with NMP at 1700 rpm and the wafer wasspin coated with 1.0% solids SE-7210 polyimide at 1700 rpm. The waferswere pre-cured at 85 C for 10 minutes, followed by a final cure at 180 Cfor 15 minutes. Polyimide surfaces were rubbed by machine with thefollowing settings:

wheel speed 343 rpm,

table speed: 3 cm/sec

non-load wheel current: 0.065 amps

loaded wheel current: 0.055 amps

left height readout: 2.530

right height readout: 2.530

The following materials were deposited in sequence from droplet onto thewafer. A two hour incubation period was used for each addition:

Protein A: 1 mg/ml

Bovine serum albumin: 0.1 mg/ml

Mouse IgG: 1 microMolar

The surface thickness of the wafer, calculated from ellipsometricreadings following each binding step, are listed below:

Polyimide coated wafer 21.0, 21.1 Protein A 23.4, 22.9, 22.9 BSA 22.3,21.8, 22.2 Mouse Antibody 24.9, 24.2These readings demonstrate the binding of Protein A to the polyimidesurface and the subsequent binding of Mouse antibody to the Protein A.

Example 12 Detection of molecular interactions on polyimide surfaces byliquid crystals

Glass slides were scrubbed with 1-Methyl-2-Pyrrolidinone(NMP), spincleaned with NMP at 1700 rpm and spin coated with 1.0% solids polyimideSE-7210 at 1700 rpm. The slides were pre-cured at 85 C for 10 min andsubjected to a final cure at 180 C for 15 minutes. The followingmaterials were added by droplet incubation in the order listed:

Protein A: 1 mg/ml

BSA 0.1 mg/ml

Mouse antibody 1 microMolar

Slides were washed with phosphate buffered saline in betweenincubations. They were dried with a stream of nitrogen and formed into acell by the addition of a top slide. Liquid crystal ZLI-1221 was addedto the space between the glass slides.

The binding of mouse antibody to the immobilized protein A present onthe polyimide surface was reported by a change in orientation of theliquid crystals. Areas with only Protein A or with Protein A plus BSAappeared dark to the naked eye when visualized through cross polars, asdid the areas of the polyimide surface which were not functionalizedwith Protein A. The areas that were functionalized with Protein A andhad been exposed to the mouse antibody, appeared white when viewedthrough a polarizing film. The contrast of areas that bound mouseantibody with non-functionalized or non-antibody exposed areas wasstrong.

This experiment has been conducted with liquid crystals ZL1-15700-000,5CB, and MLC-6710-080 with similar results to that described above.These experiments demonstrate that liquid crystals can report bindingevents hosted on functionalized polyimide surfaces.

Example 13 Demonstration of the Specificity of Binding on PolyimideSurfaces

Polyimide surfaces (SE-7210) in combination with liquid crystals can beused to specifically detect target molecules. In this experiment wedemonstrate the detection of mouse IgG and the lack of detection of ratIgG on surfaces treated with Protein A. Protein A is known to bind mouseIgG strongly while it shows a weak to no binding affinity for rat IgG.

Glass slides were scrubbed with NMP, spin cleaned with NMP at 1700 rpmand spin coated with 1.0% solids polyimide SE-7210 at 1700 rpm. Theslides were pre-cured at 85 C for 10 min and subjected to a final cureat 180 C for 15 minutes. The polyimide surface was rubbed at either ahigh pressure (2.43) or a normal pressure (2.53), or a low pressure(2.63) under standard rubbing conditions described above.

The following materials were added by droplet incubation in the orderlisted:

Protein A: 1 mg/ml

BSA 0.1 mg/ml

Mouse IgG 2a 1 microMolar

Rat IgG 1 microMolar

Slides were washed with phosphate buffered saline in betweenincubations. They were dried with a stream of nitrogen and formed into acell by the addition of a top slide. Liquid crystal ZL1-1221 was addedto the space between the glass slides.

All three rubbing pressures yielded similar results. There was verylimited disruption (the test areas appeared dark under cross polars) oncontrol samples with Protein A, Protein A+BSA or Protein A+BSA+Ratantibody. There was near total disruption on samples with ProteinA+BSA+Mouse antibody. The area appeared white under cross polars. Therewas no significant binding of the rat antibody to the Protein A. thisindicates specificity of binding on a polyimide surface.

Example 14 Polyimide Surfaces can Specifically Detect Target MoleculesUsing a “Sandwich” Technique and the Sensitivity of the System can beAdjusted by Controlling the Amount of Receptor Present on the Surface

Glass slides were coated with polyimide and rubbed using the standardprotocol described above. The slides were functionalized with thefollowing reagents, using a 30 minute incubation period for eachreagent. A series of dilutions of Protein A in the blocking agent fishgelatin of 1:99, 10:90, 25:75, and 50:50 were made to control thesensitivity of the surface to the target molecule

Protein A 1.0 mg/ml

Fish Gelatin 0.1% dilution of a 30% stock solution.

Anti-biotin 100 micrograms/ml

Biotin (100 micrograms/ml) was added to each functionalized area. Thesecondary anti-biotin antibody (100 micrograms/ml) was applied. Controlregions were included that were processed as follows:

1. Protein A+fish gelatin

2. Protein A+fish gelatin+antibody 1

3. Protein A+fish gelatin+antibody 1+biotin

Controls 1 and 2 appeared dark when viewed between cross polars,indicating that liquid crystals were uniformly aligned on the surface.Control 3 showed a minimum of disruption, but appeared mostly darkbetween cross polars, indicating uniform alignment of the liquidcrystals. The degree of disruption did appear to increase as the ratioof Protein A to fish gelatin increased, indicating an increase insensitivity of the surface. The regions exposed to the completesandwich, Protein A+fish gelatin+antibody 1+biotin+antibody 2, appearedwhite when viewed between cross polars, indicating disruption of liquidcrystal alignment and therefore the binding of the target molecule,biotin. All ratios of Protein A to fish gelatin yielded similar resultsin the complete sandwich indicating that concentrations lower than the1:99 ration could be used to tune the assay.

Example 15 Reduction of Incubation Time and Use of E7 LCF

Polyimide surfaces were prepared identically to those described inExamples 12 and 13. The reagents applied to the surface were alsoidentical to the above experiments. In this instance, the incubationtimes for each step were reduced from 2 hrs to 10 minutes. LiquidCrystal E7 was used.

A very slight increase in disruption of the liquid crystal film in thesample field was seen in comparison to the minimal disruption in theliquid crystal film over the control areas. The use of E7 significantlyreduced the response of the LC to the binding of the mouse IgG. Thenature of the disruption was very different by visual observation thanthe appearance of the disrupted liquid crystals using 5CB or ZL1-1221.

Example 16 Reduction in Incubation Time to 10 Minutes and Variation inProtein Concentration

Polyimide surfaces were prepared as described in Examples 12 and 13. Aten minute incubation time was used for each reagent. LC ZL1-1221 wasused. The concentration of the target protein (mouse antibody was varied(0.1 micromolar, 0.01 microMolar and 1.0 microMolar) Rat antibody wasused as the negative control.

Strong disruption of the liquid crystal film was observed in areasexposed to the 1.0 microMolar concentration of mouse antibody. Moderatedisruption was seen in areas exposed to the 0.1 microMolar concentrationof mouse antibody. No disruption in the liquid crystal film was observedin areas exposed to the lowest concentration (0.01 microMolar) of mouseantibody. It is possible that a ten minute incubation time under theserubbing conditions is not sufficient for sensitivity at the 0.01microMolar range.

Example 17 Variation in Protein Concentration with a 2 hr IncubationTime for Antibody

Polyimide surfaces were prepared as described in Examples 12 and 13. Allreagents are the same as used in those examples. Rat antibody is used asa negative control. LC ZL1-1221 was used. A ten minute incubation timewas used for the Protein A and BSA exposures, but a 2 hr incubation wasused for the antibody exposures.

Areas incubated with 1.0 microMolar or 0.1 microMolar mouse antibodyshowed significant disruption in the liquid crystal film. The areaincubated with 0.01 microMolar mouse antibody did not cause disruptionin the liquid crystal film. The rat antibody did not cause disruption.The number of antibodies bound to the surface area for the 0.01microMolar assay may be below the threshold needed to cause disruptionin the liquid crystal film. Sensitivity may be increased by decreasingthe functionalized surface area.

Example 18 Use of Fish Gelatin to Minimize Protein A binding sites

Four polyimide coated and rubbed slides were blotted with the materialslisted below. The slides were blotted using a 30 minute incubation timefor each of the materials. A dilution series was created using 1:99,10:90, 25:75 and 50:50 ratios of Protein A and the diluted Fish Gelatin.Each slide was blotted with one of the four mixed Protein A/Fish gelatindilution samples. Subsequent additions of primary antibody(anti-biotin), biotin, and secondary antibody (anti-biotin) were addedto the specified areas. The coating of slides and the rubbing procedureswere identical to those used in the examples above. Reagents:

Protein A: 1.0 mg/ml

Fish Gelatin: 0.1% dilution of 30% stock

Anti-biotin: 100 micrograms/ml

Biotin antigen: 100 micrograms/ml

ZL1-1221

Slight disruption was seen when the primary antibody binds to Protein Afor 50:50 and for 25:75 samples. For 10:90 ratios, the disruption isless than above and for 1:99, it is even less.

By diluting the Protein A with fish gelatin it is possible to limit theamount of primary antibody. This is an approach to limit the sensitivityof the system. This allows the rubbed polyimide to be used for systemsin which the goal is to create an assay for the detection of an antigenas well as systems for the detection of antibody. Based on theseresults, it appears that a concentration even lower that 1:99 could beused and may result in even less disruption with the primary antibodyand antigen, while still giving good disruption in response to thebinding of the secondary antibody.

Example 19 Detection of VSV-1 General Materials:

Virus: vesicular stomatitis virus-Indiana strain (VSV-I) obtained fromthe American Type Culture Collection (ATCC), Chantilly, Va., will bepropagated on BHK-21 cells. Aliquots of the initial stock will be storedat −80° C. to provide for the preparation of working stock virus. Virustiters will be calculated by plaque formation on BHK cells. Virus willbe diluted in growth medium with 10% fetal bovine serum for testing.

Antibodies: Antibodies to VSV-I will be obtained from the NationalVeterinary Diagnostic Laboratory, Ames Iowa and the ATCC, Chantilly, Va.

Polyurethane: Norland Optical Adhesive 61 (Norland Products, Cranbury,N.J. Liquid crystals: 5 CB, E7, ZLI 1221, MLC 1400-100, MLC10000-100(EMD Chemicals)

Polyimide alignment layers: SE 7210, SE 7511L (Nissan Chemicals)

Glass slides: aluminosilicate, Corning 1737 F

Design of Electrode Geometry and Fabrication of Microelectrodes

Available software such as Coulomb 3D is used to simulate the electricfield and to calculate the capacitance between in-plane electrodes.Based on the simulation results, appropriate electrode geometry(triangular, hyperbolic, interdigitated, etc.) and parameters(thickness, width, separation) will be identified so that thecapacitance between the electrodes falls within an easily detectablerange, typically in the pF range.

An order of magnitude estimate of the sensitivity of the electricaldetection system was performed. The capacitance was calculated betweeninterdigitated electrodes as shown in FIG. 3 b with 5 μm width and 5 μmspacing between them. Using a semi-infinite strip approximation, thecapacitance between electrodes with 50 fingers, each of length 500 μm,is C=0.4 ε_(eff) pF, where ε_(eff) is relative permittivity of themedium covering the electrodes. When a homogeneously aligned layer of LCundergoes orientational transition from planar to homeotropicconfiguration, the effective dielectric constant changes fromε_(planar)=38 to ε_(homeo)=8 (as calculated for the liquid crystalMLC10000-100) which corresponds to a capacitance change from 3.2 pF to15 pF. A change in capacitance in the pF range can be detected by usinga commercially available LCR meter in the laboratory setting or bymeasuring differential capacitance using an off-the-shelf chip (e.g.,MS3110 Universal Capacitive Readout from MicroSensors Inc, Costa MesaCalif.).

The sensitivity of the detection system depends on the resolution of thedevice used for measurement of the capacitance. Both commerciallyavailable LCR meters and off-the-shelf differential capacitancemeasurement chips have a resolution on the order of 0.1 fF in 10 pFlevel. An order of magnitude estimate of 0.1 fF resolution forinterdigitated electrodes described above corresponds to a total of 10virons on a 500 μm×500 μm area. This result predicts that by using anelectrical detection system it is possible to detect 10 viron particlesbound to the surface. Arrays of interdigitated co-planar electrodes aredesigned based on the calculations described above. In preferredembodiments, electrodes are in the micrometer range and fabrication ofthese electrodes will be performed by using standard photolithographicmethods of patterning followed by a lift off process. These electrodearrays are fabricated on commercially available glass substrates.

Estimation of the Change in Capacitance Between Planar and HomeotropicOrientation of Liquid Crystals

Using electrodes fabricated as described above, experimentalmeasurements are preformed to measure the capacitance of the liquidcrystal anchored on the electrodes in known orientations. Thesemeasurements are performed by coating the electrodes with LC alignmentfilms that give rise to known orientations of LCs. A thin (20 nm-thick)LC alignment layer (Nissan SE 7210) is coated onto two glass substrates(one with optimized electrodes and the other without electrodes) andbuffed to create anisotropy in the surface morphology that aligns the LCmaterial in a predetermined azimuthal direction perpendicular to theelectrode fingers. An approximately 25 μm thick optical cell isfabricated by clamping these two substrates, separated by a Mylar filmat each end, together. The liquid crystal 4-cyano-4′-pentylbiphenyl(5CB) is injected in to the gap between the substrates in isotropicphase (40° C.) and cooled down to room temperature. The capacitance ismeasured between the electrodes using a precision LCR meter (HP 42841,Agilent Technologies).

A similar cell is prepared using the homeotropic alignment layer (NissanSE 7511L) and the capacitance between the electrodes is measured. Acomparison between these two measurements provides an estimate of changein capacitance when LC undergoes orientational transition upon bindingof virus to the surface.

Preparation of the Analytic Surface

In order to provide a chemically homogeneous surface onto which toimmobilize the antibodies (for capture of virus), the co-planarelectrode arrays are coated with a thin polymeric layer. The examplesabove established that antibodies immobilized on polyurethane films havea sufficient binding capacity for detection of WNV, therefore, a thinlayer of polyurethane is spin coated onto the electrode arrays. Inparticular, a thin layer of polyurethane NOA 61 is spin coated onto thesurface and degassed in a vacuum dessicator. A planar piece ofpolydimethylsiloxane (PDMS) is placed onto the NOA 61-coated glassslide, compressed and degassed. The sandwich is cross linked for 30 minin 365 nm ultraviolet light. The PDMS is peeled from the surfaceyielding a thin 40 μm layer of polyurethane on the substrate. If the PUfilm is too thick to see any changes due to liquid crystalre-orientation, thinner layers may be produced by dilution of the NOA 61with acetone before spin coating onto the surface. The capacitancebetween the fingers of the electrodes is measured for reference.

Functionalization of the Analytic Surface and Optical Confirmation ofVirus Binding

Two procedures for immobilization of the antibodies are utilized. Bothwere found to permit detection of VSV in the examples above. The firstapproach uses the passive adsorption of antibodies onto the polyurethanesurface. The second approach employs protein A to achieve the orientedimmobilization of antibodies. The antibodies (monoclonal or rabbitpolyclonal) are allowed to adsorb to the surface from a 20 μl droplet.The VSV-binding ability of these surfaces is validated by exposing thefunctionalized areas to solutions of VSV-I at 35° C. with rocking for1.5 hours. The sample is rinsed with PBS, and 20 μm thick Mylar spacersplaced at each end of the slide. Atridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane-coated slide isplaced on top, and clamped to the bottom slide to form an optical cell.Fifteen μl of 5CB is added to the cell. The surface is then viewedbetween crossed (90°) polarizing filters. A dark field of view verifiesthe homeotropic alignment of LCs.

Example 20 Use of Capacitance Measurements in Combination with LiquidCrystals to Achieve Electrical Detection of Virus

The measurement of capacitance

Using surfaces and electrode geometries validated as described inExample 19, a high precision LCR meter is used to measure the change incapacitance between electrodes supporting a film of liquid crystal on asurface without bound virus and a film of liquid crystal upon a surfacewith bound virus. In order to determine the change in capacitanceinduced by the orientational transition, five optical cells areconstructed:

1) untreated polyurethane(PU) surface;

2) PU surface treated with antibody to VSV-I;

3) PU surface treated with antibody to VSV-I and a non-specific virussuch as herpes simplex;

4) PU surface treated with antibody to VSV-I, and VSV-I virus; and

5) PU surface treated with a non-specific antibody (e.g., anti-biotinIgG) and VSV-I virus.

A comparison of capacitance between two electrodes in all five types ofcells provides a quantitative measure of the change in capacitance uponspecific binding of VSV-1 to the surface. When implemented in the finaldevice used for assay measurements, we will employ a differentialcapacitive measurement system similar to MS3110 Universal CapacitiveReadout (MicroSensors Inc. CA) for measurements of capacitance. A systemlike the MS3110 permits facile measurement of the change in thedifferential capacitance between the electrodes treated with antibodytargeted to VSV and the control surfaces (e.g. the surfaces presentingnon-specific antibody). The output voltage of such a system is a linearfunction of the change in the differential capacitance between twoinputs fed to it. A series of virus concentrations are tested toestablish the relationship between the concentration of bound virus andthe change in capacitance of the liquid crystal film.

Selection of Optimal Liquid Crystalline Materials

It is contemplated that the sensitivity of an assay based on LCs dependupon the ability of the LCs to undergo the orientational transition froma planar to a homeotropic configuration upon binding of the virus. Thistendency to undergo the orientational transition depends on the detailedmolecular level interaction between LC molecules and the virusparticles. We will evaluate different LC materials, including 5CB, E7,MLC 1400-100, MLC10000-100, TL-205, for their applicability andeffectiveness to undergo orientational transitions. In preliminarystudies, 14 species of liquid crystals have been examined for theirresponse to lipids and have identified those that assume a homeotropicorientation in response to lipids (Table 1). The change in capacitancebetween two electrodes also depends on dielectric anisotropy of the LCmaterial, which is up to 30 for commercially available LC material.However, some reports have suggested that addition of small amounts ofpolar material could significantly increase the dielectric anisotropy ofLC materials. Accordingly, devices are constructed that include knownferroelectric materials, such as Sn₂P₂S₆ (Ouskova et al, Dielectricrelaxation spectroscopy of a nematic liquid crystal doped withferroelectric Sn₂P₂S₆ nanoparticles. Liquid Crystals 30:1-5 (2003)), asdopants for enhancement of the dielectric anisotropy of LC that willultimately increase the sensitivity of the assays.

EXAMPLE 21 Dielectrophoretic Enhancement of Mass Transport of Virus

The presence of the electrodes utilized for capacitance measurementprovides the opportunity to address a fundamental challenge thatconfronts all surface-based analytical methods. That is, the transportof the analyte from the sample matrix to the analytical surfaces isgenerally the rate-limiting step in surface-based analyses (oftenrequiring prolonged incubation times for sufficient binding to occur).Certain methods of the present invention exploit the electrodes presenton the analytical surfaces to accelerate the transport of virus to thesurface via dielectrophoresis. Thus the electrodes in the device aremultifunctional—they both increase the rate of transport of virus to thesurface (via dielectrophoresis, as described below) and form the basisof a sensitive method to report the presence of the bound virus (asdescribed in the preceding examples).

Dielectrophoresis is a phenomenon in which a polarizable particle in anon-uniform alternating current (AC) electric field experiences a netforce and moves toward the region of high or low electric fieldstrength. If the particle is more polarizable than the suspendingmedium, it moves toward the region of strong electric field and if theparticle is less polarizable than the medium, it moves toward the regionof low electric field. The magnitude of dielectrophoretic force dependsalso on, besides the dielectric properties of the particles and themedium, the gradient of the electric field and the size of the particle.For a particle of radius r_(p) in an electric field with gradientΛ/E_(rms)|² the average dielectrophoretic force is given by,

F_(DEP)=2πr_(p) ³ε_(m) Re[Ke]Λ|E _(rms)|²

where ε_(m) is the permittivity of the medium, E_(rms) is the root meansquare electric field intensity and Re[K_(E)] is the real part ofClaussius-Mossoti factor given as,

$K_{E} = \frac{\left( {ɛ_{p}^{*} - ɛ_{m}^{*}} \right)}{\left( {ɛ_{p}^{*} + {2ɛ_{m}^{*}}} \right)}$

where ε*_(p) and ε*_(m) are effective dielectric permittivity of theparticle and the medium, respectively. The direction of thedielectrophoretic force is determined by the relative sign of Re[K_(E)]which depends on the relative conductivity and permittivity of theparticle and the medium. For example, for a spherical virus particlesuspended in a physiological medium such as TSE with σ_(m)=600 mSm⁻¹,and ε_(m)=80ε₀, the single shell model yields Re[K_(E)]=−0.46 at 10 MHz.This result indicates that the virus particle will move toward theregion of lower electric field at 10 MHz. This is termed “negativedielectrophoresis”. The dieletrophoretic force exerted on a virusparticle of radius 250 nm, in a field gradient defined by hyperbolicelectrodes separated by 10 μm with AC field of strength 5 V_(pp) betweenthem, located at the edge of the electrode is approximately 3 pN. Forcomparison, the force associated with Brownian motion of the particle ina medium is of the order of F_(B)=K_(B)T/(2 r_(p)), where K_(B) is theBoltzmann constant and T is the absolute temperature. Thus at roomtemperature, the force experienced by the virus particle is on the orderof 10⁻² pN. These results clearly indicate that the dieletrophoreticforce is at least two orders of magnitude stronger than the thermalforce exerted on the particle, which is responsible for the diffusivetransport of the virus particles. Neglecting Brownian and buoyancyforces exerted on the particle, the equation of motion of the particleis determined by dielectrophoretic force and viscous drag acting on it.Using 3 pN for the dielectrophoretic force, the velocity of the virusparticle is estimated to be 700 μms⁻¹. Thus the time taken for theparticle to travel a 100 μm distance is on the order of 0.1 s. Thediffusion coefficient of the particle suspended in water can beestimated by using the Stokes-Einstein's equation; D=K_(B)T/(6πηr_(p))where η is the coefficient of viscosity of medium. The diffusioncoefficient is estimated to be 10⁻¹² m²s⁻¹. The time taken by the virusparticle to diffuse through the same distance of 100 μm in absence ofdielectrophoretic force is 1.4 hrs. This simple order of magnitudeestimate shows that the dielectrophoretic force exerted on the virusparticle suspended in a medium drives the virus particle at least fourorders of magnitude faster than by the diffusion process alone. Theseresults clearly indicate that application of the dielectrophoretic forcesignificantly enhances the mass transport of the virus particles on tothe surface, thus providing a basis for real time virus detection.

Optimization of Electrode Geometry for Dielectrophoresis of Virus

The DEP force exerted on a virus particle depends strongly on a gradientof the electric field between two electrodes. In this task, differentgeometries of electrodes, such as interdigitated, hyperbolic,rectangular, and triangular will be investigated for their ability toproduce the largest field gradient in the regions between theelectrodes. This investigation will be guided by computer modeling ofelectric fields generated by different electrode arrays. This modelingwill use commercially available software such as Coulomb 3D. FIG. 6shows the results from a simulation of hyperbolic electrodes. Theseresults show that the field gradient exhibits a local minimum at thecenter of the electrodes which will be the virus collection area fornegative dielectrophoresis.

Dielectrophoretic Transport of Virus to a Surface

Results by Morgan et al. Separation of submicron bioparticles bydielectrophoresis. Biophysical Journal 77: 516-525 (1999) indicate thatviruses exhibit both positive and negative dielectrophoresis as afunction of electric field frequency and this behavior is dependent onthe dielectric properties of the suspending medium. This cross-overeffect in the frequency domain is investigated by using differentsuspension media for the virus particles. Buffers, such as Tris-salineEDTA, phosphate buffered saline, growth media such as Minimum Essentialmedia with 10% fetal calf sera as well as various animal and human seraand standard viral transport media will be examined for their dielectricproperties and their effects on the dielectrophoretic force exerted onthe virus. Combinations of variables such as strength and frequency ofthe applied AC field, and ionic strength of the suspending medium, thatyield the maximum response in the least amount of time and with theleast amount of sample will be identified. To monitor these events inreal time the virus particles are labeled. A method recently publishedby Akin et al., Real-time virus trapping and fluorescent imaging inmicrofluidic devices, Nano Letters 4: 257-259 (2004) is utilized whichused a lipophilic carbocyanin dyes to label the envelope of vacciniavirus. The dyes (DiOC63 and DIL, Molecular Probes, Calif.) label thelipid membrane and capsid proteins of the virus and permittedvisualization of surface bound vaccinia by digital epifluorescencemicroscopy at 400× magnification.

Antibody functionalized surfaces with integrated microelectrodes (FIG.3) fabricated as described above are exposed to a 20 μL droplet oflabeled VSV-1. An AC electric field is applied across the electrodes toinduce dielectrophoretic motion of the virus particles to the surface inthe region between the electrodes. VSV-1 binds to the antibodies on thesurface. The field is turned off and the unbound virus is washed fromthe surface. These events are monitored in real time using aZeiss-Axiovert 200 M microscope.

The optimal parameters for the dielectrophoresis of VSV-1 are identifiedby observation of the intensity of fluorescence seen within the centerregion of the electric field. The intensity of the fluorescence iscorrelated with the known concentrations of virus in the applied sample.The strength and frequency of the applied field that permit detection ofthe least amount of virus in under 5 minutes are chosen for testingagainst various suspension media of physiologic ionic strength. Suchmedia include typical clinical materials such as serum, nasal swabfluid, and viral transport media to determine how the strength andfrequency required for viral transport will be affected by these commonsample fluids.

Example 22 Use of Beads for Capture of an Analyte Followed byNon-Specific Detection

Preparation of beads. Sera-Mag beads (0.8M in diameter) werefunctionalized with either 0.4 mg/mL EDC (Aldrich) or 1.1 mg/mLSulfo-NHS (Pierce). First, 27 μl of 5% Sera-Mag beads were diluted in 1mL of the functionalizing agent. Reactions were carried out 15 minutesand then quenched with 2-mercaptoethanol. The beads were washed 3 timeswith 25 mM MES and 37.5 mM NaCl. The washed beads were centrifuged at11,300 rpm for 5 minutes. Removal of buffer was followed by the additionof fresh buffer. Next, 0.06 μM aF1pAb (100 μg/mL) was added for 1.5-2hours during which the beads were rotated and mixed. The beads werequenched for amine by adding a final concentration of 10 mMD-glucosamine. The beads were then washed in PBS+D-glucusoamine (10 mM)for 20 minutes. The beads were transferred to regular microfuge tubesblocked with BSA to prevent non-specific binding of non-target moleculesto the beads.

Detection of F1 antigen from Yersinia pestis. Magnetic beads werefunctionalized with anti-F1 polyclonal antibody as described above. Abinding solution was prepared from PBS, Tween 0.05%, Heparin 50 μg/mL,and 100 mM additional NaCl. Wash solutions were prepared from PBS/Tween0.05% and PBS/Tween 0.05%+200 mM additional NaCl. The beads werefunctionalized and stored in PBS at 0.135% w/v, the same concentrationat which the beads were utilized. In the experiment, 1 mL of beadsolution was utilized and bound up to 5 μg/ml of F1 at over 99.0%. AnELISA on F1 samples applied before and after capture on the on the beadswas carried out. The F1 samples were placed on ELISA strip wellsmeasured for levels of F1 protein using anti-F1 polyclonal antibody. Thetwo conditions, start material and depleted material, were then graphedagainst absorbance 450 nm as result of ELISA colorimetric assay. Thisallowed to estimation of % of F1 captured by the functionalized beads.The data showed that almost all the F1 was absorbed to the beads. The F1antigen was then eluted to a detection substrate surface comprisingpolyimide. The polyimide surface is prepared as follows: Undilutedpolyimide 7511 liquid is applied to a slide, spin coated, pre-cured at80° C. for 15 minutes, cured at 180° C. for one hour and stored at RT.Further, in the last step of the protocol, 10 uL 0.1M pH 2.4 glycine ismixed with the particle beads, and the tubes are then vortexed in amicrofuge and placed on a magnetic stand to obtain separation of beadsfrom supernatant. The low pH glycine serves to elute bound F1 and itsantibody complex from the bead surface. 10 uL of the eluent is appliedto the polyimide surface for the LC assay. Following elution, an opticalcell was formed by adding a second substrate separated from thedetection substrate by a thin mylar film. Mesogens (5CB) were thenintroduced into the optical cell.

Surfaces functionalized with polyimide 7511L orient liquid crystals in ahomeotropic alignment in the absence of bound analyte. When analyte ispresent, the homeotropic orientation is disrupted. When viewed throughcross polar lenses, homeotropic orientation is indicated by a darkfield, while disrupted orientation is indicated by a bright field, asshown in FIG. 8. A digitized image taken with two Polaroid filters atcross polar configuration at 0°. Polyimide 7511L slides were preparedfrom 40% undiluted stock. These slides, in absence of protein, assumehomeotropic alignment as shown in dark background. Upon protein binding,homeotropic alignment is disrupted and shown in white background. In thetop row, from left to right, are elution samples from aF1pAb beadsinitially blocked with RNase A treated with BSA 400 ng/mL, F1 500 pg/mLand 2.5 ng/mL. The bottom row is 25, 100, and 400 ng/mL concentrationsof F1. After initial protein binding in binding solution PBS/T/H/N,aF1pAb beads were washed in PBS/Tween 0.05%, 200 mM additional NaCl(PBS/T/N) wash solution.

An additional example demonstrates how this approach one can determinepresence and quantity of the target molecule. FIG. 9 below demonstratesthat with increasing F1 concentration, the disruption of homeotropicalignment increases as indicated by increased white signal againstaligned LC black background on homeotropic aligning polyimide surfaces.This type of assay can be used to quantify levels of the F1 antigen.

In FIG. 9, the digitized figures were taken by polarized microscope withcross polar filters (0°). Polyimide 7511L slides were prepared from 40%undiluted stock. In absence of protein, these slides assume homeotropicalignment as shown in by the dark background. Upon protein bindinghomeotropic alignment is disrupted (indicated by a white background).The following samples were analyzed: elution buffer, BSA (200 ng/mL),and F1 elutions of beads exposed to 5, 10, 50 and 200 ng/mLconcentrations of F1. The conditions were as described above. Theseexperiments were carried in triplicate and FIG. 9 had intermediatelevels of signal with respect to two other replicates (not shown). Ascan be seen, it was possible to detect 5 ng/mL analyte.

FIG. 10 is a graphic representation of luminosity index for theexperiment depicted in FIG. 9. These experiments were carried intriplicate and figure chosen above have intermediate levels of signalwith respect to two other replicates. Elution buffer was also applied toone area and used as reference for Luminosity index shown in the graphbelow.

In further experiments, a rubbed polyimide surface was utilized. FIG. 11shows a digitized image taken with a polarized microscope with crosspolar filters (0 degrees). Polyimide SE 7210 1.5% slides were rubbed at2.55 ml/m setting at 4.0 cm/s table speed with a wheel speed of 343 rpm.FIG. 11 shows the results of elutions to the polyimide surface fromfunctionalized aF1pAb beads that were treated with variousconcentrations of F1 or BSA as control. After washing, the beads weretreated by the addition of rabbit anti-mouse IgG (5 μg/mL) andanti-mouse FC IgG (6 μg/mL). The complex was eluted off of the beadswith 10 μl acid elution of 0.1 M glycine pH 2.3. Elution samples wereneutralized by addition of 1 μl 1 M Tris pH 7.5. The samples werecontacted with the polyimide substrate and an optical cell wasconstructed by placing a mylar spacer on the substrate clamping anothersubstrate onto the first with bulldog clamp. 5CB was applied in liquidphase and after cooling the digitized image was acquired.

FIG. 12 provides the results of further experiments demonstrating thedetection of an analyte (F1) from a complex medium (chicken serum). Inthese experiments, chicken serum (CS) was diluted to 20% with thebinding solution PBS/T/H/N. BSA blocked beads functionalized as abovewere used. Six 0.5 mL suspensions of aF1pAb beads (0.135% concentration)were contacted with the diluted CS. After binding, all tubes were washedwith PBS/Tween buffer. The beads were then washed a second time with 100μL 0.5×PBS+2 mM SDS or 100 μL 0.1Mglycine pH 4.0. After mixing, the washbuffers were removed by pelleting the beads in PBS/Tween 0.05%. Thebeads were then washed with PBS/Tween/NaCl (additional 200 mM). aF1mAbat 3 μg/mL was then added to the beads in 0.5 ml tubes. The reactionswere mixed and the supernatant was removed. Next, 0.75 mL of 5 μg/ml ofboth RAM and anti-mouse FC IgG were added for 5 minutes. The beads werewashed with PBS/Tween/NaCl and then with PBS. Elution buffer (10 μL) wasadded to each tube mixed by vortexing for 3-4 minutes. The elutionbuffer (2 μL aliquots) was the applied to a 7511L homeotropic polyimideslide. The slides were placed on a heated plate for 2-3 minutes untildroplets have evaporated. The slides were then washed with dH₂O anddried with gas nitrogen. An optical cell was constructed by using abulldog clamp to clamp together the treated substrate and an anothersubstrate with a mylar spacer in between. 5CB in nematic phase wasapplied at room temperature. After 5 minutes, the homeotropic liquidcrystal was observed.

FIG. 12 is a digitized image taken with cross polar filters (0 degrees).In top row are elutions from beads treated contacted with 20% CS with noadditional wash, SDS wash, and a 0.1M glycine pH 4.0 wash. In the lowerrow are elutions from beads contacted with 20% CS+F1 50 ng/mL with noadditional wash, SDS wash, or a 0.1M glycine pH 4.0 wash. FIG. 13presents the results of the same experiments as analyzed with apolarized microscope. The digitized images were taken by polarizedmicroscope where the polaroid filters are cross polar at 0°. The orderof the treatments is the same as for FIG. 12.

The SDS and 0.1M Glycine washes were conducted to remove the BSA blockerfrom the functionalized beads so that there be no BSA signal interferingwith F1 detection. As can be seen in FIG. 12 and FIG. 13 (polarizedmicroscope image), both the SDS and Glycine washes eliminated the signalfrom the control top row second and third spots (20% Chicken Serum).Since the functionalized beads were blocked with BSA, it is most likelythe BSA blocker that creates the signal seen in the first spot.

Example 23 Homeotropic Orientation of Liquid Crystals by Cells

Tables 2 and 3 present the results of experiments in which differentliquid crystals were surveyed for their ability to be homeotropicallyoriented by cultured cells. Many liquid crystals align homeotropicallyin response to phospholipids and cholesterol. Phospholipids (2 ul; 0.01M in chloroform) were applied to discrete marked areas on glass slides.The phospholipids had dioleoyl alkyl chains and the followingheadgroups: phosphatidylserine (DOPS), phosphatidylglycerol (DOPG),phosphatidylethanolamine (DOPE), phosphatidylserine (DOPS), phosphatidicacid (DOPA), and

TABLE 2 Survey of liquid crystals for alignment by cells and by slideexposed to medium. Liquid Crystal FBS/DMEM 3T3 cells 4OCB DisruptedHomeotropic 5CB Disrupted Homeotropic 6CHBT Planar, with defectsHomeotropic E7 Disrupted Homeotropic ZLI-1221 Planar, streaky DisruptedZLI-1557 Planar with streaky defects Homeotropic ZLI-2222 Planar, minordefects Homeotropic ZLI-3225 Planar with streaky defects Homeotropic(tilt) ZLI-3497 Planar with streaky defects Homeotropic (tilt) ZLI-4431Planar with streaky defects Homeotropic (tilt) ZLI-4446 Planar, withdefects Homeotropic ZLI-5070 Planar with streaky defects Homeotropic(tilt) MLC-6080 Planar with squiggly defects Homeotropic MLC-6466 Planarwith streaky defects Homeotropic MLC-6710-080 Planar with streakydefects Homeotropic MLC-15700-000 Planar, streaky Homeotropic TL205Somewhat planar Homeotropiclysophosphatidylcholine (DOLPC). After the solvent dried, optical cellswere assembled with liquid crystals applied nematically and heated toisotropy. Homeotropic alignment was confirmed by conoscopic analysis.Chol=cholesterol; C=cholesteric alignment; Bkg=background alignment;U=unaligned; H=homeotropically aligned; ND indicates not done due tobackground. 4OCB, 4′-octyl-4-biphenyl-carbonitrile (Aldrich); 6CHBT,1-(trans-4-hexylcyclohexyl)-4-isothiocyanato-benzene. All other liquidcrystals are from EM Industries/Merck.

TABLE 3 Investigation of phospholipid influence on liquid crystalalignment. Liquid Crystal Bkg DOPS DOPG DOPC DOPE DOPA DOLPC Chol 4OCB HND ND ND ND ND ND ND 5CB U H H H Planar H U 6CHBT U H H H H H U E7Twisted H H H Twisted H H planar planar ZLI-1221 H H H H H H H ZLI-1557H H H H H H H ZLI-2222 H H H H H H H ZLI-3225 U H H H H H H ZLI-3497 HND ND ND ND ND ND ND ZLI-4431 Chol U U U U U U U ZLI-4446 H ND ND ND NDND ND ND ZLI-5070 Twisted H H H H H H planar MLC-6080 U H H H H H HMLC-6466 U H H H H H H MLC-6710-080 U H H H H H H MLC-15700-000 H H H UH H U TL205 U H H H H H H

Example 24 Detection of Analyte with Tagged Lipids

This experiment describes the detection of binding of liposomes labeledwith biotin to the immobilized anti-biotin antibody using liquidcrystals. Anti-biotin immobilized glass substrates were prepared asfollows. PrecisionCT slides (Bioslide Technologies; Cat# BSP-SC02-C)cleaned in plasma asher (20 min, 275 watts, 200 millitor) were immersedin 2% APES (3-Aminopropyltriethoxysilane; Pierce) in dry acetone for 2min. Slides were transferred to pure acetone and stirred for 5 min andrinsed with acetone to remove excess of silane. Slides were dried withnitrogen and kept at 110° C. oven for 45 min. Slides were removed fromoven and after they reached room temperature areas were marked on oneside of slide. 1 mg/ml BS3 (Bis (Sulfosuccinimidyl) suberate; Pierce)cross-linker was applied as 10 ul drops on marked areas and incubatedfor 15 min at room temperature. After rinsing excess cross-linker withwater surfaces were dried with nitrogen gas. 100 ug/ml and 20 ug/mlanti-biotin antibody (Sigma; Anti-Biotin Developed in Goat) was appliedon BS3 treated surfaces and incubated for 2 hrs at room temperature or 1hr at room temperature and later transferred to 4° C. overnight.Antibody immobilized surfaces were rinsed with milliQ water to removeunbound protein and dried with N2. Liposome labeled with biotin (17.84micromol phospholipid/ml) and unlabeled liposome (18.7 micromolphospholipid/ml) were diluted 100fold with PBS buffer, and 10 ul ofliposome was applied on anti-biotin treated surface. PBS buffer wasadded on one area as buffer control. After 1:30 hrs incubation at roomtemperature, surfaces was rinsed with water and N2 dried. Two proteintreated surfaces were kept apart by inserting 20 μm mylar spacer at twosides and two surfaces were aligned in anti-parallel direction. Twosurfaces were held together by using bulldog clips placed along themylar placed sides. The cells were heated to ˜40° C. by placing them onhot plate. I also used hot air to warm the air around the cells. 5CB washeated into its isotropic phase within a glass syringe. A drop of 5CBwas drawn into the cavity between two surfaces by capillary force. Oncefilled with 5CB, the cell was removed form hot plate. After reachingroom temperature, isotropic phase of 5CB transformed to the nematicstate. Optical images were taken at crossed polar position usingpolarizing microscope. The above experiment was done in triplicates. Thedata from one experiment is presented in FIG. 15.

Binding of biotin labeled liposome to the immobilized anti-biotinantibody changes disrupted surface to homeotropic (black) where as nochange was noticed with unlabeled liposome. This experiment demonstratesthe use of liposomes labeled with target molecule to studyreceptor-ligand interactions.

The biotin labeled liposomes were prepared from 100 micromol (75 mg) eggphosphatidylcholine; 1 micromol (1.28 mg)phosphatidylethanolamine-N-(lissamine rhodamine B; sulfonyl) ammoniumsalt; 1 micromol (1.05 mg)1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(cap Biotinyl) sodiumsalt. The control liposomes were prepared from 100 micromol (75 mg) eggphosphatidylcholine; 1 micromol (1.28 mg);phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) ammoniumsalt

The lipid mixtures were prepared by combining the desired quantities ofthe components in chloroform solution. Each solution was then divided intwo for liposome preparation. For each batch The solution was dried downin a B24/40 boiling tube on a rotary evaporator. The resultant film wassuspended in 5 ml. of hydrated isopropylether. To the lipid solution wasadded 2 ml of buffer. At this stage the buffer does not mix with theether phase. The tube was stoppered, and the mixture was sonicated in abath type sonicator (Laboratory Supply company, Hicksville N.Y.) at 55 Cto give a water-in-ether emulsion. The ether was then evaporated fromthe emulsion using a rotary evaporator, bath temperature 55 C. Afterether removal, the mixture formed a viscous gel, which on agitation,broke down to give a liposome suspension. After liposome formation, thetwo 50 micromol batches for sample and control liposomes respectivelywere combined for the next step.

The size of the liposomes was then reduced to a smaller and more uniformdiameter by a process known as extrusion, wherein they were passedthrough a series of polycarbonate membranes of defined pore size. Theextrusion chamber was heated to 6° C., and the liposomes were passedfive times through a 0.4 micron pore size polycarbonate membrane, andthen five times through a 0.1 micron pore size polycarbonate membrane.The process of extrusion noticeably reduces the turbidity of thesuspension.

The liposomes were then dialysed overnight at 4 C against 1 liter of thesuspension buffer so as to eliminate any residual traces ofisopropylether. The liposomes were analyzed for their phospholipidcontent using the phosphorus assay of Bartlett, and were found tocontain:

Sample (biotin) liposomes: 17.84 micromol phospholipid/ml

Control liposomes: 18.7 micromol phospholipid/ml

The liposomes were analyzed for their size using a Nicomp 380 particlesizer. The volume-weighted gaussian mean diameters were:

Sample (biotin) liposomes: 134 nanometer

Control liposomes: 157 nanometer.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in organicchemistry, materials science, chemical engineering, virology, biology,genetics, or related fields are intended to be within the scope of thefollowing claims.

1. A method for detecting a biological entity containing a lipid moietycomprising: a) providing: i) a sample suspected of containing a lipidmoiety; ii) a detection device comprising a substrate comprising atleast one detection region; iii) mesogens; b) contacting said detectionregion with said sample; c) contacting said substrate with saidmesogens, wherein the presence of said biological entity containing alipid moiety is indicated by a change in said mesogens over saiddetection regions.
 2. The method of claim 1, wherein said change in saidmesogens is selected from the group consisting of a change in color, achange in texture, a change in tilt, and change in orientation.
 3. Themethod of claim 1, wherein said change in mesogens is detected by amethod selected from the group consisting of visual detection, opticaldetection, spectroscopic detection, light transmission, and electricaldetection.
 4. The method of claim 1, wherein said lipid moiety is aconstituent of an entity selected from the group consisting of aeukaryotic cell, a bacterium, a Mycoplasma, a virus, and a liposome orcombinations thereof.
 5. The method of claim 4, wherein said liposomefurther displays a ligand.
 6. The method of claim 1, wherein saidsubstrate is selected from the group consisting of metal films, glass,silicon, diamond and polymeric materials.
 7. The method of claim 6,wherein said polymeric materials are selected from the group consistingof polyurethane, PDMS, polyimide, polystyrene, polycarbonate andpolyisocyanoacrylate.
 8. The method of claim 1, wherein said mesogen isselected from the group consisting of 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butlyaniline and combinations thereof.
 9. Themethod of claim 1, wherein said detection region further comprises arecognition moiety that recognizes said biological entity.
 10. Themethod of claim 1, wherein said substrate comprises a plurality ofdetection regions.
 11. The method of claim 1, wherein said change insaid mesogens is detected by viewing said detection device between crosspolar lenses.
 12. The method of claim 1, wherein said sample is selectedfrom the group consisting of biological fluids, tissue homogenates,feces, vesicular fluids, tissue swabs, and media in which a biologicalagents have been cultured or prepared.
 13. The method of claim 12,wherein said biological fluid is selected from the group consisting ofcerebral-spinal fluid, urine, serum, plasma, nasal secretions, sputum,semen and saliva.
 14. A method for detecting an analyte comprising: a)providing: i. a sample suspected of containing an analyte ii. arecognition moiety that is complexed with a lipid, iii. mesogens b)contacting said sample with said recognition moiety-lipid complexwherein said recognition moiety interacts with said analyte, to form ananalyte-recognition moiety-lipid complex; c) contacting saidanalyte-recognition moiety-lipid complex with said mesogens, wherein thepresence of said analyte is indicated by a change in said mesogens. 15.The method of claim 14, wherein said mesogens are homeotropicallyoriented in the presence of said analyte-recognitionmoiety-lipid-complex.
 16. The method of claim 14, wherein said change ofsaid mesogens is detected by a method selected from the group consistingof visual detection, optical detection, spectroscopic detection, lighttransmission, and electrical detection.
 17. The method of claim 14,wherein said sample is selected from the group consisting of biologicalfluids, tissue homogenates, feces, vesicular fluids, tissue swabs, andmedia in which biological agents have been cultured or prepared.
 18. Themethod of claim 17, wherein said biological fluid is selected from thegroup consisting of cerebral-spinal fluid, urine, serum, plasma, nasalsecretions, sputum, semen and saliva.
 19. The method of claim 14,wherein said sample is applied to a substrate.
 20. The method of claim19, wherein said substrate is selected from the group consisting ofmetal films, glass, silicon, diamond and polymeric materials.
 21. Themethod of claim 19, wherein said substrate further comprises at leastone detection region.
 22. The method of claim 21, wherein said detectionregion further comprises a ligand that recognizes said biologicalentity.
 23. The method of claim 14, wherein said recognitionmoiety-lipid complex is immobilized on said substrate.
 24. The method ofclaim 23, wherein said recognition moiety is an enzymatic substrate. 25.The method of claim 14 wherein said analyte is an enzyme.
 26. The methodof claim 19, wherein said substrate comprises a plurality of detectionregions.
 27. The method of claim 20, wherein said polymeric materialsare selected from the group consisting of polyurethane, PDMS, polyimide,polystyrene, polycarbonate and polyisocyanoacrylate.
 28. The method ofclaim 14, wherein said mesogen is selected from the group consisting of4-cyano-4′-pentylbiphenyl, N-(4-methoxybenzylidene)-4-butlyaniline andcombinations thereof.